Msln targeting trispecific proteins and methods of use

ABSTRACT

Provided herein are mesothelin (MSLN) targeting trispecific proteins comprising a domain binding to CD3, a half-life extension domain, and a domain binding to MSLN. Also provided are pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such MSLN targeting trispecific proteins. Also disclosed are methods of using the disclosed MSLN targeting trispecific proteins in the prevention, and/or treatment of diseases, conditions and disorders.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application Nos.62/505,747 filed on May 12, 2017, and 62/657,434 filed Apr. 13, 2018,each incorporated by reference herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 11, 2018, isnamed 47517-720_201_SL.txt and is 293,251 bytes in size.

BACKGROUND OF THE INVENTION

The selective destruction of an individual cell or a specific cell typeis often desirable in a variety of clinical settings. For example, it isa primary goal of cancer therapy to specifically destroy tumor cells,while leaving healthy cells and tissues intact and undamaged. One suchmethod is by inducing an immune response against the tumor, to makeimmune effector cells such as natural killer (NK) cells or cytotoxic Tlymphocytes (CTLs) attack and destroy tumor cells.

Mesothelin (MSLN) is a GPI-linked membrane bound tumor antigen MSLN isoverexpressed ovarian, pancreatic, lung and triple-negative breastcancers and mesothelioma. Normal tissue expression of MSLN is restrictedto single-cell, mesothelial layers lining the pleural, pericardial, andperitoneal cavities. Overexpression of MSLN is associated with poorprognosis in lung adenocarcinoma and triple-negative breast cancer. MSLNhas been used as cancer antigen for numerous modalities, includingimmunotoxins, vaccines, antibody drug conjugates and CAR-T cells. Earlysigns of clinical efficacy have validated MSLN as a target, buttherapies with improved efficacy are needed to treat MSLN-expressingcancers.

SUMMARY OF THE INVENTION

One embodiment provides a mesothelin binding trispecific protein,wherein said protein comprises

(a) a first domain (A) which specifically binds to human CD3;(b) a second domain (B) which is a half-life extension domain; and(c) a third domain (C) which specifically binds to MSLN,wherein the domains are linked in the order H₂N-(A)-(C)—(B)—COOH,H₂N—(B)-(A)-(C)—COOH, H₂N—(C)—(B)-(A)-COOH, or by linkers L1 and L2. Insome embodiments, the first domain comprises a variable light domain anda variable heavy domain each of which is capable of specifically bindingto human CD3. In some embodiments, the first domain is humanized orhuman. In some embodiments, the second domain binds albumin. In someembodiments, the second domain comprises a scFv, a variable heavy domain(VH), a variable light domain (VL), a peptide, a ligand, or a smallmolecule. In some embodiments, the third domain comprises a VHH domain,a scFv, a VH domain, a VL domain, a non-Ig domain, a ligand, a knottin,or a small molecule entity that specifically binds to MSLN. In someembodiments, the third domain comprises a VHH domain. In someembodiments, said VHH domain comprises one or more conserved regionscomprising a sequence identical to or comprising one or more amino acidresidue substitutions relative to SEQ ID NO: 41, 42, 43, or 44. In someembodiments, said VHH domain comprises a conserved region comprising asequence identical to or comprising one or more amino acid residuesubstitutions relative to SEQ ID NO: 41. In some embodiments, said VHHdomain comprises a conserved region comprising a sequence identical toor comprising one or more amino acid residue substitutions relative toSEQ ID NO: 42. In some embodiments, said VHH domain comprises aconserved domain comprising a sequence identical to or comprising one ormore amino acid residue substitutions relative to SEQ ID NO: 43. In someembodiments, said VHH domain comprises a conserved domain comprising asequence identical to or comprising one or more amino acid residuesubstitutions relative to SEQ ID NO: 44. In some embodiments, said VHHdomain comprises (i) a stretch of amino acids corresponding to SEQ IDNO: 41; (ii) a stretch of amino acids corresponding to SEQ ID NO: 42;(iii) a stretch of amino acids corresponding to SEQ ID NO: 43, and (iv)a stretch of amino acids corresponding to SEQ ID NO: 44. In someembodiments, said humanized VHH domain comprises the following formula:f1-r1-f2-r2-f3-r3-f4 wherein, r1 is identical to or comprises one ormore amino acid residue substitutions relative to any one of SEQ IDNos.: 134-44; r2 is identical to or comprises one or more amino acidresidue substitutions relative to any one of SEQ ID Nos.: 173-183; andr3 is identical to or comprises one or more amino acid residuesubstitutions relative to SEQ ID Nos.: 212-222; and wherein f1, f2, f3and f4 are framework residues.

In some embodiments, said VHH domain comprises the following formula:

f1-r1-f2-r2-f3-r3-f4

wherein, r1 is identical to or comprises one or more amino acid residuesubstitutions relative to SEQ ID NO: 51; r2 is identical to or comprisesone or more amino acid residue substitutions relative to SEQ ID NO: 52;and r3 is identical to or comprises one or more amino acid residuesubstitutions relative to SEQ ID NO: 53; and wherein f1, f2, f3 and f4are framework residues. In some embodiments, said VHH domain comprises asequence that is at least 80% identical to a sequence selected from thegroup consisting of SEQ ID NOs: 1-29. In some embodiments, the thirddomain comprises selected sequence from the group consisting of SEQ IDNOs: 1-29. In some embodiments, the third domain is a humanized VHHdomain. In some embodiments, said humanized VHH domain comprises one ormore conserved regions comprising a sequence identical to or comprisingone or more amino acid residue substitutions relative to SEQ ID NO: 45,46, 47, 48, 49, or 50. In some embodiments, said VHH domain comprises aconserved region comprising a sequence identical to or comprising one ormore amino acid residue substitutions relative to SEQ ID NO: 45. In someembodiments, said VHH domain comprises a conserved region comprising asequence identical to or comprising one or more amino acid residuesubstitutions relative to SEQ ID NO: 46. In some embodiments, said VHHdomain comprises a conserved domain comprising a sequence identical toor comprising one or more amino acid residue substitutions relative toSEQ ID NO: 47. In some embodiments, said VHH domain comprises aconserved domain comprising a sequence identical to or comprising one ormore amino acid residue substitutions relative to SEQ ID NO: 48. In someembodiments, said VHH domain comprises a conserved domain comprising asequence identical to or comprising one or more amino acid residuesubstitutions relative to SEQ ID NO: 49. In some embodiments, said VHHdomain comprises a conserved domain comprising a sequence identical toor comprising one or more amino acid residue substitutions relative toSEQ ID NO: 50. In some embodiments, said VHH domain comprises (i) astretch of amino acids corresponding to SEQ ID NO: 45, (ii) a stretch ofamino acids corresponding to SEQ ID NO: 46, (iii) a stretch of aminoacids corresponding to SEQ ID NO: 47, (iv) a stretch of amino acidscorresponding to SEQ ID NO: 48, (v) a stretch of amino acidscorresponding to SEQ ID NO: 49, and (vi) a stretch of amino acidscorresponding to SEQ ID NO: 50. In some embodiments, said humanized VHHdomain comprises the following formula:

f1-r1-f2-r2-f3-r3-f4

wherein, r1 is identical to or comprises one or more amino acid residuesubstitutions relative to SEQ ID NO: 54; r2 is identical to or comprisesone or more amino acid residue substitutions relative to SEQ ID NO: 55;and r3 is identical to or comprises one or more amino acid residuesubstitutions relative to SEQ ID NO: 56; and wherein f1, f2, f3 and f4are framework residues. In some embodiments, said third domain comprisesa CDR1 sequence comprising a sequence as set forth in any one of SEQ IDNos.: 51, 54, and 106-144. In some embodiments, said third domaincomprises a CDR2 sequence comprising a sequence as set forth in any oneof SEQ ID Nos.: 52, 55, and 145-183. In some embodiments, said thirddomain comprises a CDR2 sequence comprising a sequence as set forth inany one of SEQ ID Nos.: 53, 56, and 184-222. One embodiment provides, amesothelin (MSLN) binding trispecific protein, comprising the sequenceas set forth in SEQ ID NO: 98.

In some embodiments, the third domain comprises a sequence selected fromthe group consisting of SEQ ID NOs: 30-40, and 102-105. In someembodiments, the third domain binds to a human mesothelin proteincomprising the sequence set forth as SEQ ID NO: 57. In some embodiments,the third domain binds to an epitope of mesothelin, wherein said epitopeis located in region I, comprising amino acid residues 296-390 of SEQ IDNO: 57, region II comprising amino acid residue 391-486 of SEQ ID NO:57, or region III comprising amino acid residues 487-598 of SEQ ID NO:57. In some embodiments, linkers L1 and L2 are each independentlyselected from (GS)_(n) (SEQ ID NO: 87), (GGS)_(n) (SEQ ID NO: 88),(GGGS)_(n) (SEQ ID NO: 89), (GGSG)_(n) (SEQ ID NO: 90), (GGSGG)_(n) (SEQID NO: 91), or (GGGGS)_(n) (SEQ ID NO: 92), wherein n is 1, 2, 3, 4, 5,6, 7, 8, 9, or 10. In some embodiments, linkers L1 and L2 are eachindependently (GGGGS)₄ (SEQ ID NO: 95) or (GGGGS)₃ (SEQ ID NO: 96). Insome embodiments, the domains are linked in the orderH₂N—(C)—(B)-(A)-COOH. In some embodiments, the protein is less thanabout 80 kDa. In some embodiments, the protein is about 50 to about 75kDa. In some embodiments, the protein is less than about 60 kDa. In someembodiments, the protein has an elimination half-time of at least about50 hours. In some embodiments, the protein has an elimination half-timeof at least about 100 hours. In some embodiments, the protein hasincreased tissue penetration as compared to an IgG to the same MSLN. Insome embodiments, the protein comprises a sequence selected from thegroup consisting of SEQ ID NO: 58-86, 98, 100, and 101. One embodimentprovides a mesothelin binding trispecific protein, comprising thesequence as set forth in SEQ ID NO: 98. One embodiment provides amesothelin binding trispecific protein, wherein said protein comprises:(a) a first domain (A) which specifically binds to human CD3; (b) asecond domain (B) which is a half-life extension domain; and (c) a thirddomain (C) which specifically binds to MSLN, wherein the domains arelinked in the order H₂N-(A)-(C)—(B)—COOH, H₂N—(B)-(A)-(C)—COOH,H₂N—(C)—(B)-(A)-COOH, or by linkers L1 and L2, wherein said third domaincomprises one or more CDRs selected from the group consisting of SEQ IDNos.: 51-56 and 106-222. In some embodiments, said third domaincomprises a CDR1 comprising a sequence as set forth in any one of SEQ IDNos.: 51, 54, and 106-144. In some embodiments, said third domaincomprises a CDR2 comprising a sequence as set forth in any one of SEQ IDNos.: 52, 55, and 145-183. In some embodiments, said third domaincomprises a CDR2 comprising a sequence as set forth in any one of SEQ IDNos.: 53, 56, and 184-222. In some embodiments, said third domaincomprises a framework region 1 (f1) comprising a sequence as set forthin any one of SEQ ID Nos.: 262-300. In some embodiments, said thirddomain comprises a framework region (f2) sequence as set forth in anyone of SEQ ID Nos.: 301-339. In some embodiments, said third domaincomprises a framework region (f3) a sequence as set forth in any one ofSEQ ID Nos.: 340-378. In some embodiments, the protein comprises asequence selected from the group consisting of SEQ ID NOs: 58-86, 98,100, and 101. In some embodiments, the protein comprises a sequence asset forth in SEQ ID NO: 98.

One embodiment provides a pharmaceutical composition comprising (i) theMSLN binding trispecific protein according to any one of the aboveembodiments and (ii) a pharmaceutically acceptable carrier.

One embodiment provides a process for the production of a mesothelinbinding trispecific protein according to any one of the aboveembodiments, said process comprising culturing a host transformed ortransfected with a vector comprising a nucleic acid sequence encoding amesothelin binding trispecific protein according to any one of the aboveembodiments under conditions allowing the expression of the mesothelinbinding trispecific protein and recovering and purifying the producedprotein from the culture.

One embodiment provides a method for the treatment or amelioration of aproliferative disease, or a tumorous disease, comprising theadministration of the mesothelin binding trispecific protein accordingto any one of the above embodiments, to a subject in need thereof. Oneembodiment provides a method for the treatment or amelioration of aproliferative disease, or a tumorous disease, comprising administrationof a mesothelin binding trispecific protein comprising a sequence as setforth in any one of SEQ ID Nos.: 58-86, 98, 100, and 101.

In some embodiments, the subject is human. In some embodiments, themethod further comprises administration of an agent in combination withthe single domain mesothelin binding protein according to any one of theabove embodiments. In some embodiments, the mesothelin bindingtrispecific protein selectively binds to tumor cells expressingmesothelin. In some embodiments, the mesothelin binding trispecificprotein mediates T cell killing of tumor cells expressing mesothelin. Insome embodiments, the tumorous disease comprises a solid tumor disease.In some embodiments, the solid tumor disease comprises mesothelioma,lung cancer, gastric cancer, ovarian cancer, or triple negative breastcancer. In some embodiments, the solid tumor disease is metastatic.

One embodiment provides a method for the treatment or amelioration of aproliferative disease, or a tumorous disease, comprising administrationof a mesothelin binding trispecific protein comprising a sequenceselected from the group consisting of SEQ ID NOs: 58-86, 98, 100, and101. In some embodiments, the mesothelin binding trispecific proteinselectively binds to tumor cells expressing mesothelin. In someembodiments, the mesothelin binding trispecific protein directs T cellkilling of tumor cells expressing mesothelin. In some embodiments, thetumorous disease comprises a solid tumor disease. In some embodiments,the solid tumor disease comprises mesothelioma, lung cancer, gastriccancer, ovarian cancer, or triple negative breast cancer. In someembodiments, the solid tumor disease is metastatic.

One embodiment provides a method for the treatment or amelioration of aproliferative disease, or a tumorous disease, comprising administrationof a mesothelin binding trispecific protein comprising a sequence as setforth in SEQ ID NO: 98. In some embodiments, the method comprisesadministering the mesothelin binding trispecific protein at a dose of upto 10 mg/kg. In some embodiments, the protein is administered once aweek. In some embodiments, the protein is administered twice per week.In some embodiments, the protein is administered every other week. Insome embodiments, the protein is administered every three weeks.

One embodiment provides a method for the treatment or amelioration of aproliferative disease, or a tumorous disease, comprising administrationof a mesothelin binding trispecific protein comprising a sequence as setforth in any one of SEQ ID NOs: 58-86, 98, 100, and 101. In someembodiments, the method comprises administering the mesothelin bindingtrispecific protein at a dose of up to 10 mg/kg. In some embodiments,the protein is administered once a week. In some embodiments, theprotein is administered twice per week. In some embodiments, the proteinis administered every other week. In some embodiments, the protein isadministered every three weeks.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is schematic representation of an exemplary MSLN targetingtrispecific antigen-binding protein where the protein has an constantcore element comprising an anti-CD3c single chain variable fragment(scFv) and an anti-ALB variable heavy chain region; and an anti-MSLNbinding domain that can be a VHH, a VH, scFv, a non-Ig binder, or aligand.

FIG. 2 illustrates the effectivity of exemplary TriTAC molecules (2A2and 2A4) in killing of OVCAR8 cells that expresses the target proteinMSLN.

FIG. 3 illustrates that an exemplary TriTAC molecule of this disclosure(MH6T TriTAC) was able to direct T cells from five donors (donor 02;donor 86; donor 41; donor 81; and donor 35) to kill Caov3 cells. Thefigure also illustrates that a control TriTAC molecule (GFP TriTAC) wasnot able to direct T cells from the five donors (donor 02; donor 86;donor 41; donor 81; and donor 35) to kill Caov3 cells.

FIG. 4 illustrates that an exemplary TriTAC molecule of this disclosure(MH6T TriTAC) was able to direct T cells from five donors (donor 02;donor 86; donor 41; donor 81; and donor 35) to kill OVCAR3 cells. Thefigure also illustrates that a control TriTAC molecule (GFP TriTAC) wasnot able to direct T cells from the five donors (donor 02; donor 86;donor 41; donor 81; and donor 35) to kill OVCAR3 cells.

FIG. 5 illustrates that an exemplary TriTAC molecule of this disclosure(MH6T TriTAC) was able to direct T cells from a healthy donor to killcells that express MSLN (OVCAR3 cells; Caov4 cells; OVCAR3 cells; andOVCAR8 cells). The figure also illustrates that the MH6T TriTAC was notable to direct T cells from the healthy donor to kill cells that do notexpress MSLN (MDAPCa2b cells; and NCI-H510A cells).

FIG. 6 illustrates that an exemplary TriTAC molecule of this disclosure(MH6T TriTAC) was able to direct T cells from cynomolgus monkeys to killhuman ovarian cancer cell line (OVCAR3 cells; Caov3 cells). The figurealso illustrates that a control TriTAC molecule (GFP TriTAC) was notable to direct the T cells from cynomolgus monkeys to kill human ovariancancer cells lines (OVCAR3 cells; Caov3 cells).

FIG. 7 illustrates that an exemplary TriTAC molecule of this disclosure(MH6T TriTAC) was able to direct killing of MSLN expressing NCI-H2052mesothelioma cells by T cells, in the presence or absence of human serumalbumin (HSA).

FIG. 8 illustrates that an exemplary TriTAC molecule of this disclosure(MH6T TriTAC) was able to activate T cells from four healthy donors(donor 2; donor 86; donor 35; and donor 81), as demonstrated bysecretion of TNF-α from the T cells, in presence of the MH6T TriTAC andMSLN-expressing Caov4 cells.

FIG. 9 illustrates that an exemplary TriTAC molecule of this disclosure(MH6T TriTAC) was able to activate T cells from four healthy donors(donor 2; donor 86; donor 35; and donor 81), as demonstrated byactivation of CD69 expression on the T cells, in presence of the MH6TTriTAC and MSLN-expressing OVCAR8 cells.

FIG. 10A and FIG. 10B illustrate binding of an exemplary TriTAC moleculeof this disclosure (MH6T TriTAC) to MSLN expressing cell lines or MSLNnon-expressing cell lines. FIG. 10A shows binding of the MH6T TriTACwith MSLN expressing cells (Caov3 cells—top left panel; Caov4 cells—topright panel; OVCAR3 cells—bottom left panel; OVCAR8 cells—bottom rightpanel); and further illustrates lack of binding of a control TriTAC (GFPTriTAC) to the same cell lines. FIG. 10B shows lack of binding of boththe MH6T TriTAC and the GFP TriTAC to MSLN non-expressing cell lines(MDCA2b cells—left panel; NCI-H510A cells—right panel).

FIG. 11 illustrates binding of an exemplary TriTAC molecule of thisdisclosure (MH6T TriTAC) to T cells from four healthy donors (donor2-top left panel; donor 35-top right panel; donor 41-bottom left panel;donor 81-bottom right panel).

FIG. 12 illustrates that an exemplary TriTAC molecule of this disclosure(MH6T TriTAC) was able to inhibit tumor growth in NCG mice implantedwith MSLN expressing NCI-H292 cells.

FIG. 13 illustrates pharmacokinetic profile of an exemplary TriTACmolecule of this disclosure, MH6T TriTAC. Serum levels of the MH6TTriTAC molecule, at various time points following injection into twocynomolgus monkeys, are shown in the plot.

FIG. 14 shows results from binding affinity measurements of twoexemplary trispecific molecules of this disclosure, TriTAC 74 and TriTAC75, and EC₅₀ values for killing of SKOV3 and OVCAR cells by the twoTriTAC molecules.

FIG. 15 illustrates pharmacokinetic profiles of two exemplary TriTACmolecules of this disclosure, TriTAC 75 and TriTAC 74. Serum levels ofthe TriTAC molecules, at various time points following injection intocynomolgus monkeys, are shown in the plot.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are trispecific proteins that target mesothelin (MSLN),pharmaceutical compositions thereof, as well as nucleic acids,recombinant expression vectors and host cells for making such proteinsthereof. Also provided are methods of using the disclosed MSLN targetingtrispecific proteins in the prevention, and/or treatment of diseases,conditions and disorders. The MSLN targeting trispecific proteins arecapable of specifically binding to MSLN as well as CD3 and have ahalf-life extension domain, such as a domain binding to human albumin(ALB). FIG. 1 depicts one non-limiting example of a trispecificMSLN-binding protein.

In one aspect, the MSLN targeting trispecific proteins comprise a domain(A) which specifically binds to CD3, a domain (B) which specificallybinds to human albumin (ALB), and a domain (C) which specifically bindsto MSLN. The three domains in MSLN targeting trispecific proteins arearranged in any order. Thus, it is contemplated that the domain order ofthe MSLN targeting trispecific proteins are:

H₂N-(A)-(B)—(C)—COOH,

H₂N-(A)-(C)—(B)—COOH,

H₂N—(B)-(A)-(C)—COOH,

H₂N—(B)—(C)-(A)-COOH,

H₂N—(C)—(B)-(A)-COOH, or

H₂N—(C)-(A)-(B)—COOH.

In some embodiments, the MSLN targeting trispecific proteins have adomain order of H₂N-(A)-(B)—(C)—COOH. In some embodiments, the MSLNtargeting trispecific proteins have a domain order ofH₂N-(A)-(C)—(B)—COOH. In some embodiments, the MSLN targetingtrispecific proteins have a domain order of H₂N—(B)-(A)-(C)—COOH. Insome embodiments, the MSLN targeting trispecific proteins have a domainorder of H₂N—(B)—(C)-(A)-COOH. In some embodiments, the MSLN targetingtrispecific proteins have a domain order of H₂N—(C)—(B)-(A)-COOH. Insome embodiments, the MSLN targeting trispecific proteins have a domainorder of H₂N—(C)-(A)-(B)—COOH.

In some embodiments, the MSLN targeting trispecific proteins have theHSA binding domain as the middle domain, such that the domain order isH₂N-(A)-(B)—(C)—COOH or H₂N—(C)—(B)-(A)-COOH. It is contemplated that insuch embodiments where the ALB binding domain as the middle domain, theCD3 and MSLN binding domains are afforded additional flexibility to bindto their respective targets.

In some embodiments, the MSLN targeting trispecific proteins describedherein comprise a polypeptide having a sequence described in Table 1(SEQ ID NO: 58-86, 98, 100, and 101) and subsequences thereof. In someembodiments, the trispecific antigen binding protein comprises apolypeptide having at least 70%-95% or more homology to a sequencedescribed in Table 1 (SEQ ID NO: 58-86, 98, 100 and 101). In someembodiments, the trispecific antigen binding protein comprises apolypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or morehomology to a sequence described in Table 1 (SEQ ID NO: 58-86, 98, 100and 101).

The MSLN targeting trispecific proteins described herein are designed toallow specific targeting of cells expressing MSLN by recruitingcytotoxic T cells. This improves efficacy compared to ADCC (antibodydependent cell-mediated cytotoxicity), which is using full lengthantibodies directed to a sole antigen and is not capable of directlyrecruiting cytotoxic T cells. In contrast, by engaging CD3 moleculesexpressed specifically on these cells, the MSLN targeting trispecificproteins can crosslink cytotoxic T cells with cells expressing MSLN in ahighly specific fashion, thereby directing the cytotoxic potential ofthe T cell towards the target cell. The MSLN targeting trispecificproteins described herein engage cytotoxic T cells via binding to thesurface-expressed CD3 proteins, which form part of the TCR. Simultaneousbinding of several MSLN trispecific antigen-binding protein to CD3 andto MSLN expressed on the surface of particular cells causes T cellactivation and mediates the subsequent lysis of the particular MSLNexpressing cell. Thus, MSLN targeting trispecific proteins arecontemplated to display strong, specific and efficient target cellkilling. In some embodiments, the MSLN targeting trispecific proteinsdescribed herein stimulate target cell killing by cytotoxic T cells toeliminate pathogenic cells (e.g., tumor cells expressing MSLN). In someof such embodiments, cells are eliminated selectively, thereby reducingthe potential for toxic side effects.

The MSLN targeting trispecific proteins described herein confer furthertherapeutic advantages over traditional monoclonal antibodies and othersmaller bispecific molecules. Generally, the effectiveness ofrecombinant protein pharmaceuticals depends heavily on the intrinsicpharmacokinetics of the protein itself. One such benefit here is thatthe MSLN targeting trispecific proteins described herein have extendedpharmacokinetic elimination half-time due to having a half-lifeextension domain such as a domain specific to HSA. In this respect, theMSLN targeting trispecific proteins described herein have an extendedserum elimination half-time of about two, three, about five, aboutseven, about 10, about 12, or about 14 days in some embodiments. Thiscontrasts to other binding proteins such as BiTE or DART molecules whichhave relatively much shorter elimination half-times. For example, theBiTE CD19×CD3 bispecific scFv-scFv fusion molecule requires continuousintravenous infusion (i.v.) drug delivery due to its short eliminationhalf-time. The longer intrinsic half-times of the MSLN targetingtrispecific proteins solve this issue thereby allowing for increasedtherapeutic potential such as low-dose pharmaceutical formulations,decreased periodic administration and/or novel pharmaceuticalcompositions.

The MSLN targeting trispecific proteins described herein also have anoptimal size for enhanced tissue penetration and tissue distribution.Larger sizes limit or prevent penetration or distribution of the proteinin the target tissues. The MSLN targeting trispecific proteins describedherein avoid this by having a small size that allows enhanced tissuepenetration and distribution. Accordingly, the MSLN targetingtrispecific proteins described herein, in some embodiments have a sizeof about 50 kD to about 80 kD, about 50 kD to about 75 kD, about 50 kDto about 70 kD, or about 50 kD to about 65 kD. Thus, the size of theMSLN targeting trispecific proteins is advantageous over IgG antibodieswhich are about 150 kD and the BiTE and DART diabody molecules which areabout 55 kD but are not half-life extended and therefore cleared quicklythrough the kidney.

In further embodiments, the MSLN targeting trispecific proteinsdescribed herein have an optimal size for enhanced tissue penetrationand distribution. In these embodiments, the MSLN targeting trispecificproteins are constructed to be as small as possible, while retainingspecificity toward its targets. Accordingly, in these embodiments, theMSLN targeting trispecific proteins described herein have a size ofabout 20 kD to about 40 kD or about 25 kD to about 35 kD to about 40 kD,to about 45 kD, to about 50 kD, to about 55 kD, to about 60 kD, to about65 kD. In some embodiments, the MSLN targeting trispecific proteinsdescribed herein have a size of about 50 kD, 49, kD, 48 kD, 47 kD, 46kD, 45 kD, 44 kD, 43 kD, 42 kD, 41 kD, 40 kD, about 39 kD, about 38 kD,about 37 kD, about 36 kD, about 35 kD, about 34 kD, about 33 kD, about32 kD, about 31 kD, about 30 kD, about 29 kD, about 28 kD, about 27 kD,about 26 kD, about 25 kD, about 24 kD, about 23 kD, about 22 kD, about21 kD, or about 20 kD. An exemplary approach to the small size isthrough the use of single domain antibody (sdAb) fragments for each ofthe domains. For example, a particular MSLN trispecific antigen-bindingprotein has an anti-CD3 sdAb, anti-ALB sdAb and an sdAb for MSLN. Thisreduces the size of the exemplary MSLN trispecific antigen-bindingprotein to under 60 kD. Thus in some embodiments, the domains of theMSLN targeting trispecific proteins are all single domain antibody(sdAb) fragments. In other embodiments, the MSLN targeting trispecificproteins described herein comprise small molecule entity (SME) bindersfor ALB and/or the MSLN. SME binders are small molecules averaging about500 to 2000 Da in size and are attached to the MSLN targetingtrispecific proteins by known methods, such as sortase ligation orconjugation. In these instances, one of the domains of MSLN trispecificantigen-binding protein is a sortase recognition sequence, e.g., LPETG(SEQ ID NO: 97). To attach a SME binder to MSLN trispecificantigen-binding protein with a sortase recognition sequence, the proteinis incubated with a sortase and a SME binder whereby the sortaseattaches the SME binder to the recognition sequence. Known SME bindersinclude MIP-1072 and MIP-1095 which bind to mesothelin. In yet otherembodiments, the domain which binds to MSLN of MSLN targetingtrispecific proteins described herein comprise a knottin peptide forbinding MSLN. Knottins are disufide-stabilized peptides with a cysteineknot scaffold and have average sizes about 3.5 kD. Knottins have beencontemplated for binding to certain tumor molecules such as MSLN. Infurther embodiments, domain which binds to MSLN of MSLN targetingtrispecific proteins described herein comprise a natural MSLN ligand.

Another feature of the MSLN targeting trispecific proteins describedherein is that they are of a single-polypeptide design with flexiblelinkage of their domains. This allows for facile production andmanufacturing of the MSLN targeting trispecific proteins as they can beencoded by single cDNA molecule to be easily incorporated into a vector.Further, because the MSLN targeting trispecific proteins describedherein are a monomeric single polypeptide chain, there are no chainpairing issues or a requirement for dimerization. It is contemplatedthat the MSLN targeting trispecific proteins described herein have areduced tendency to aggregate unlike other reported molecules such asbispecific proteins with Fc-gamma immunoglobulin domains.

In the MSLN targeting trispecific proteins described herein, the domainsare linked by internal linkers L1 and L2, where L1 links the first andsecond domain of the MSLN targeting trispecific proteins and L2 linksthe second and third domains of the MSLN targeting trispecific proteins.Linkers L1 and L2 have an optimized length and/or amino acidcomposition. In some embodiments, linkers L1 and L2 are the same lengthand amino acid composition. In other embodiments, L1 and L2 aredifferent. In certain embodiments, internal linkers L1 and/or L2 are“short”, i.e., consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12amino acid residues. Thus, in certain instances, the internal linkersconsist of about 12 or less amino acid residues. In the case of 0 aminoacid residues, the internal linker is a peptide bond. In certainembodiments, internal linkers L1 and/or L2 are “long”, i.e., consist of15, 20 or 25 amino acid residues. In some embodiments, these internallinkers consist of about 3 to about 15, for example 8, 9 or 10contiguous amino acid residues. Regarding the amino acid composition ofthe internal linkers L1 and L2, peptides are selected with propertiesthat confer flexibility to the MSLN targeting trispecific proteins, donot interfere with the binding domains as well as resist cleavage fromproteases. For example, glycine and serine residues generally provideprotease resistance. Examples of internal linkers suitable for linkingthe domains in the MSLN targeting trispecific proteins include but arenot limited to (GS)_(n)(SEQ ID NO: 87), (GGS)_(n) (SEQ ID NO: 88),(GGGS)_(n)(SEQ ID NO: 89), (GGSG)_(n) (SEQ ID NO: 90), (GGSGG)_(n) (SEQID NO: 91), (GGGGS)_(n) (SEQ ID NO: 92), (GGGGG)_(n) (SEQ ID NO: 93), or(GGG)_(n)(SEQ ID NO: 94), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.In one embodiment, internal linker L1 and/or L2 is (GGGGS)₄ (SEQ ID NO:95) or (GGGGS)₃ (SEQ ID NO: 96).

CD3 Binding Domain

The specificity of the response of T cells is mediated by therecognition of antigen (displayed in context of a majorhistocompatibility complex, MHC) by the TCR. As part of the TCR, CD3 isa protein complex that includes a CD3γ (gamma) chain, a CD3δ (delta)chain, and two CD3ε (epsilon) chains which are present on the cellsurface. CD3 associates with the α (alpha) and β (beta) chains of theTCR as well as CD3 ζ (zeta) altogether to comprise the complete TCR.Clustering of CD3 on T cells, such as by immobilized anti-CD3 antibodiesleads to T cell activation similar to the engagement of the T cellreceptor but independent of its clone-typical specificity.

In one aspect, the MSLN targeting trispecific proteins described hereincomprise a domain which specifically binds to CD3. In one aspect, theMSLN targeting trispecific proteins described herein comprise a domainwhich specifically binds to human CD3. In some embodiments, the MSLNtargeting trispecific proteins described herein comprise a domain whichspecifically binds to CD3γ. In some embodiments, the MSLN targetingtrispecific proteins described herein comprise a domain whichspecifically binds to CD36. In some embodiments, the MSLN targetingtrispecific proteins described herein comprise a domain whichspecifically binds to CD3ε.

In further embodiments, the MSLN targeting trispecific proteinsdescribed herein comprise a domain which specifically binds to the TCR.In certain instances, the MSLN targeting trispecific proteins describedherein comprise a domain which specifically binds the α chain of theTCR. In certain instances, the MSLN targeting trispecific proteinsdescribed herein comprise a domain which specifically binds the β chainof the TCR.

In certain embodiments, the CD3 binding domain of the MSLN targetingtrispecific proteins described herein exhibit not only potent CD3binding affinities with human CD3, but show also excellentcrossreactivity with the respective cynomolgus monkey CD3 proteins. Insome instances, the CD3 binding domain of the MSLN targeting trispecificproteins are cross-reactive with CD3 from cynomolgus monkey. In certaininstances, human:cynomolgous K_(D) ratios for CD3 are between 5 and 0.2.

In some embodiments, the CD3 binding domain of the MSLN trispecificantigen-binding protein can be any domain that binds to CD3 includingbut not limited to domains from a monoclonal antibody, a polyclonalantibody, a recombinant antibody, a human antibody, a humanizedantibody. In some instances, it is beneficial for the CD3 binding domainto be derived from the same species in which the MSLN trispecificantigen-binding protein will ultimately be used in. For example, for usein humans, it may be beneficial for the CD3 binding domain of the MSLNtrispecific antigen-binding protein to comprise human or humanizedresidues from the antigen binding domain of an antibody or antibodyfragment.

Thus, in one aspect, the antigen-binding domain comprises a humanized orhuman antibody or an antibody fragment, or a murine antibody or antibodyfragment. In one embodiment, the humanized or human anti-CD3 bindingdomain comprises one or more (e.g., all three) light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) of a humanized or human anti-CD3 binding domain describedherein, and/or one or more (e.g., all three) heavy chain complementarydetermining region 1 (HC CDR1), heavy chain complementary determiningregion 2 (HC CDR2), and heavy chain complementary determining region 3(HC CDR3) of a humanized or human anti-CD3 binding domain describedherein, e.g., a humanized or human anti-CD3 binding domain comprisingone or more, e.g., all three, LC CDRs and one or more, e.g., all three,HC CDRs.

In some embodiments, the humanized or human anti-CD3 binding domaincomprises a humanized or human light chain variable region specific toCD3 where the light chain variable region specific to CD3 compriseshuman or non-human light chain CDRs in a human light chain frameworkregion. In certain instances, the light chain framework region is a λ(lamda) light chain framework. In other instances, the light chainframework region is a κ (kappa) light chain framework.

In some embodiments, the humanized or human anti-CD3 binding domaincomprises a humanized or human heavy chain variable region specific toCD3 where the heavy chain variable region specific to CD3 compriseshuman or non-human heavy chain CDRs in a human heavy chain frameworkregion.

In certain instances, the complementary determining regions of the heavychain and/or the light chain are derived from known anti-CD3 antibodies,such as, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4),teplizumab (MGA031), visilizumab (Nuvion), SP34, TR-66 or X35-3, VIT3,BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2,TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6,T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31.

In one embodiment, the anti-CD3 binding domain is a single chainvariable fragment (scFv) comprising a light chain and a heavy chain ofan amino acid sequence provided herein. As used herein, “single chainvariable fragment” or “scFv” refers to an antibody fragment comprising avariable region of a light chain and at least one antibody fragmentcomprising a variable region of a heavy chain, wherein the light andheavy chain variable regions are contiguously linked via a shortflexible polypeptide linker, and capable of being expressed as a singlepolypeptide chain, and wherein the scFv retains the specificity of theintact antibody from which it is derived. In an embodiment, the anti-CD3binding domain comprises: a light chain variable region comprising anamino acid sequence having at least one, two or three modifications(e.g., substitutions) but not more than 30, 20 or 10 modifications(e.g., substitutions) of an amino acid sequence of a light chainvariable region provided herein, or a sequence with 95-99% identity withan amino acid sequence provided herein; and/or a heavy chain variableregion comprising an amino acid sequence having at least one, two orthree modifications (e.g., substitutions) but not more than 30, 20 or 10modifications (e.g., substitutions) of an amino acid sequence of a heavychain variable region provided herein, or a sequence with 95-99%identity to an amino acid sequence provided herein. In one embodiment,the humanized or human anti-CD3 binding domain is a scFv, and a lightchain variable region comprising an amino acid sequence describedherein, is attached to a heavy chain variable region comprising an aminoacid sequence described herein, via a scFv linker. The light chainvariable region and heavy chain variable region of a scFv can be, e.g.,in any of the following orientations: light chain variable region-scFvlinker-heavy chain variable region or heavy chain variable region-scFvlinker-light chain variable region.

In some instances, scFvs which bind to CD3 are prepared according toknown methods. For example, scFv molecules can be produced by linking VHand VL regions together using flexible polypeptide linkers. The scFvmolecules comprise a scFv linker (e.g., a Ser-Gly linker) with anoptimized length and/or amino acid composition. Accordingly, in someembodiments, the length of the scFv linker is such that the VH or VLdomain can associate intermolecularly with the other variable domain toform the CD3 binding site. In certain embodiments, such scFv linkers are“short”, i.e. consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12amino acid residues. Thus, in certain instances, the scFv linkersconsist of about 12 or less amino acid residues. In the case of 0 aminoacid residues, the scFv linker is a peptide bond. In some embodiments,these scFv linkers consist of about 3 to about 15, for example 8, 9 or10 contiguous amino acid residues. Regarding the amino acid compositionof the scFv linkers, peptides are selected that confer flexibility, donot interfere with the variable domains as well as allow inter-chainfolding to bring the two variable domains together to form a functionalCD3 binding site. For example, scFv linkers comprising glycine andserine residues generally provide protease resistance. In someembodiments, linkers in a scFv comprise glycine and serine residues. Theamino acid sequence of the scFv linkers can be optimized, for example,by phage-display methods to improve the CD3 binding and production yieldof the scFv. Examples of peptide scFv linkers suitable for linking avariable light domain and a variable heavy domain in a scFv include butare not limited to (GS)_(n) (SEQ ID NO: 87), (GGS)_(n)(SEQ ID NO: 88),(GGGS)_(n)(SEQ ID NO: 89), (GGSG)_(n)(SEQ ID NO: 90), (GGSGG)_(n) (SEQID NO: 91), (GGGGS)_(n) (SEQ ID NO: 92), (GGGGG)_(n) (SEQ ID NO: 93), or(GGG)_(n)(SEQ ID NO: 94), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.In one embodiment, the scFv linker can be (GGGGS)₄ (SEQ ID NO: 95) or(GGGGS)₃ (SEQ ID NO: 96). Variation in the linker length may retain orenhance activity, giving rise to superior efficacy in activity studies.

In some embodiments, CD3 binding domain of MSLN trispecificantigen-binding protein has an affinity to CD3 on CD3 expressing cellswith a K_(D) of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nMor less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5nM or less, 1 nM or less, or 0.5 nM or less. In some embodiments, theCD3 binding domain of MSLN trispecific antigen-binding protein has anaffinity to CD3ε, γ, or δ with a K_(D) of 1000 nM or less, 500 nM orless, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM orless. In further embodiments, CD3 binding domain of MSLN trispecificantigen-binding protein has low affinity to CD3, i.e., about 100 nM orgreater.

The affinity to bind to CD3 can be determined, for example, by theability of the MSLN trispecific antigen-binding protein itself or itsCD3 binding domain to bind to CD3 coated on an assay plate; displayed ona microbial cell surface; in solution; etc. The binding activity of theMSLN trispecific antigen-binding protein itself or its CD3 bindingdomain of the present disclosure to CD3 can be assayed by immobilizingthe ligand (e.g., CD3) or the MSLN trispecific antigen-binding proteinitself or its CD3 binding domain, to a bead, substrate, cell, etc.Agents can be added in an appropriate buffer and the binding partnersincubated for a period of time at a given temperature. After washes toremove unbound material, the bound protein can be released with, forexample, SDS, buffers with a high pH, and the like and analyzed, forexample, by Surface Plasmon Resonance (SPR).

Half-Life Extension Domain

Contemplated herein are domains which extend the half-life of anantigen-binding domain. Such domains are contemplated to include but arenot limited to Albumin binding domains, Fc domains, small molecules, andother half-life extension domains known in the art.

Human albumin (ALB) (molecular mass □67 kDa) is the most abundantprotein in plasma, present at about 50 mg/ml (600 μM), and has ahalf-life of around 20 days in humans. ALB serves to maintain plasma pH,contributes to colloidal blood pressure, functions as carrier of manymetabolites and fatty acids, and serves as a major drug transportprotein in plasma.

Noncovalent association with albumin extends the elimination half-timeof short lived proteins. For example, a recombinant fusion of an albuminbinding domain to a Fab fragment resulted in an in vivo clearance of 25-and 58-fold and a half-life extension of 26- and 37-fold whenadministered intravenously to mice and rabbits respectively as comparedto the administration of the Fab fragment alone. In another example,when insulin is acylated with fatty acids to promote association withalbumin, a protracted effect was observed when injected subcutaneouslyin rabbits or pigs. Together, these studies demonstrate a linkagebetween albumin binding and prolonged action.

In one aspect, the MSLN targeting trispecific proteins described hereincomprise a half-life extension domain, for example a domain whichspecifically binds to ALB. In some embodiments, the ALB binding domainof MSLN trispecific antigen-binding protein can be any domain that bindsto ALB including but not limited to domains from a monoclonal antibody,a polyclonal antibody, a recombinant antibody, a human antibody, ahumanized antibody. In some embodiments, the ALB binding domain is asingle chain variable fragments (scFv), single-domain antibody such as aheavy chain variable domain (VH), a light chain variable domain (VL) anda variable domain (VHH) of camelid derived single domain antibody,peptide, ligand or small molecule entity specific for HSA. In certainembodiments, the ALB binding domain is a single-domain antibody. Inother embodiments, the HSA binding domain is a peptide. In furtherembodiments, the HSA binding domain is a small molecule. It iscontemplated that the HSA binding domain of MSLN trispecificantigen-binding protein is fairly small and no more than 25 kD, no morethan 20 kD, no more than 15 kD, or no more than 10 kD in someembodiments. In certain instances, the ALB binding is 5 kD or less if itis a peptide or small molecule entity.

The half-life extension domain of MSLN trispecific antigen-bindingprotein provides for altered pharmacodynamics and pharmacokinetics ofthe MSLN trispecific antigen-binding protein itself. As above, thehalf-life extension domain extends the elimination half-time. Thehalf-life extension domain also alters pharmacodynamic propertiesincluding alteration of tissue distribution, penetration, and diffusionof the trispecific antigen-binding protein. In some embodiments, thehalf-life extension domain provides for improved tissue (includingtumor) targeting, tissue distribution, tissue penetration, diffusionwithin the tissue, and enhanced efficacy as compared with a proteinwithout an half-life extension domain. In one embodiment, therapeuticmethods effectively and efficiently utilize a reduced amount of thetrispecific antigen-binding protein, resulting in reduced side effects,such as reduced non-tumor cell cytotoxicity.

Further, the binding affinity of the half-life extension domain can beselected so as to target a specific elimination half-time in aparticular trispecific antigen-binding protein. Thus, in someembodiments, the half-life extension domain has a high binding affinity.In other embodiments, the half-life extension domain has a mediumbinding affinity. In yet other embodiments, the half-life extensiondomain has a low or marginal binding affinity. Exemplary bindingaffinities include KD concentrations at 10 nM or less (high), between 10nM and 100 nM (medium), and greater than 100 nM (low). As above, bindingaffinities to ALB are determined by known methods such as SurfacePlasmon Resonance (SPR).

In some embodiments, ALB binding domains described herein comprise asingle domain antibody.

Mesothelin (MSLN) Binding Domain

Mesothelin is a glycoprotein present on the surface of cells of themesothelial lining of the peritoneal, pleural and pericardial bodycavities. The mesothelin gene (MSLN) encodes a 71 kD precursor proteinthat is processed to a 40 kD protein termed mesothelin, which is aglycosyl-phosphatidylinositol-anchored glycoprotein present on the cellsurface (Chang, et al, Proc Natl Acad Sci USA (1996) 93:136-40). Themesothelin cDNA was cloned from a library prepared from the HPC-Y5 cellline (Kojima et al. (1995) J. Biol. Chem. 270:21984-21990). The cDNAalso was cloned using the monoclonal antibody K1, which recognizesmesotheliomas (Chang and Pastan (1996) Proc. Natl. Acad. Sci. USA93:136-40). Mesothelin is a differentiation antigen whose expression innormal human tissues is limited to mesothelial cells lining the bodycavity, such as the pleura, pericardium and peritoneum. Mesothelin isalso highly expressed in several different human cancers, includingmesotheliomas, pancreatic adenocarcinomas, ovarian cancers, stomach andlung adenocarcinomas. (Hassan, et al., Eur J Cancer (2008) 44:46-53)(Ordonez, Am J Surg Pathol (2003) 27:1418-28; Ho, et al., Clin CancerRes (2007) 13:1571-5). Mesothelin is overexpressed in a vast majority ofprimary pancreatic adenocarcinomas with rare and weak expression seen inbenign pancreatic tissue. Argani P, et al. Clin Cancer Res. 2001;7(12):3862-3868. Epithelial malignant pleural mesothelioma (MPM)universally expresses mesothelin while sarcomatoid MPM likely does notexpress mesothelin. Most serous epithelial ovarian carcinomas, and therelated primary peritoneal carcinomas, express mesothelin.

Mesothelin can also be used a marker for diagnosis and prognosis ofcertain types of cancer because trace amounts of mesothelin can bedetected in the blood of some patients with mesothelin-positive cancers(Cristaudo et al., Clin. Cancer Res. 13:5076-5081, 2007). It has beenreported that mesothelin may be released into serum through deletion atits carboxyl terminus or by proteolytic cleavage from its membrane boundform (Hassan et al., Clin. Cancer Res. 10:3937-3942, 2004). An increasein the soluble form of mesothelin was detectable several years beforemalignant mesotheliomas occurred among workers exposed to asbestos(Creaney and Robinson, Hematol. Oncol. Clin. North Am. 19:1025-1040,2005). Furthermore, patients with ovarian, pancreatic, and lung cancersalso have elevated soluble mesothelin in serum (Cristaudo et al., Clin.Cancer Res. 13:5076-5081, 2007; Hassan et al., Clin. Cancer Res.12:447-453, 2006; Croso et al., Cancer Detect. Prev. 30:180-187, 2006).Accordingly, mesothelin is an appropriate target for methods of diseaseprevention or treatment and there is a need for effective antibodiesspecific for mesothelin.

It has been shown that cell surface mature mesothelin comprises threedistinct domains, namely Regions I (comprising residues 296-390), II(comprising residues 391-486), and III (comprising residue 487-598).(Tang et al., A human single-domain antibody elicits potent antitumoractivity by targeting an epitope in mesothelin close to the cancer cellsurface, Mol. Can. Therapeutics, 12(4): 416-426, 2013). The firstantibodies generated against mesothelin for therapeutic interventionwere designed to interfere with the interaction between mesothelin andCA-125. Phage display identified the Fv SS, which was affinity optimizedand used to generate a recombinant immunotoxin targeting mesothelin,SS1P. The MORAb-009 antibody amatuximab, which also uses SS1, recognizesa non-linear epitope in the amino terminal 64 amino acids of mesothelin,within region I. The SS1 Fv was also used to generate chimeric antigenreceptor-engineered T cells. Recently, new anti-mesothelin antibodieshave been reported that recognize other regions of the mesothelinprotein.

There is still a need for having available further options for thetreatment of solid tumor diseases related to the overexpression ofmesothelin, such as ovarian cancer, pancreatic cancer, mesothelioma,lung cancer, gastric cancer and triple negative breast cancer. Thepresent disclosure provides, in certain embodiments, MSLN targetingtrispecific proteins containing binding domains which specifically bindto MSLN on the surface of tumor target cells.

The design of the MSLN targeting trispecific proteins described hereinallows the binding domain to MSLN to be flexible in that the bindingdomain to MSLN can be any type of binding domain, including but notlimited to, domains from a monoclonal antibody, a polyclonal antibody, arecombinant antibody, a human antibody, a humanized antibody. In someembodiments, the binding domain to MSLN is a single chain variablefragments (scFv), single-domain antibody such as a heavy chain variabledomain (VH), a light chain variable domain (VL) and a variable domain(VHH) of camelid derived single domain antibody. In other embodiments,the binding domain to MSLN is a non-Ig binding domain, i.e., antibodymimetic, such as anticalins, affilins, affibody molecules, affimers,affitins, alphabodies, avimers, DARPins, fynomers, kunitz domainpeptides, and monobodies. In further embodiments, the binding domain toMSLN is a ligand or peptide that binds to or associates with MSLN. Inyet further embodiments, the binding domain to MSLN is a knottin. In yetfurther embodiments, the binding domain to MSLN is a small molecularentity.

In some embodiments, the MSLN binding domain binds to a proteincomprising the sequence of SEQ ID NO: 57. In some embodiments, the MSLNbinding domain binds to a protein comprising a truncated sequencecompared to SEQ ID NO: 57.

In some embodiments, the MSLN binding domains disclosed herein recognizefull-length mesothelin. In certain instances, the MSLN binding domainsdisclosed herein recognize an epitope in region I (comprising amino acidresidues 296-390 of SEQ ID NO: 57), region II (comprising amino acidresidue 391-486 of SEQ ID NO: 57), or region III (comprising amino acidresidues 487-598 of SEQ ID NO: 57) of mesothelin. It is contemplatedthat the MSLN binding domains of the present disclosure may, in someembodiments, recognize and bind to epitopes that are located outsideregions I, II, or III of mesothelin. In yet other embodiments aredisclosed MSLN binding domains that recognize and bind to an epitopedifferent than the MORAb-009 antibody.

In some embodiments, the MSLN binding domain is an anti-MSLN antibody oran antibody variant. As used herein, the term “antibody variant” refersto variants and derivatives of an antibody described herein. In certainembodiments, amino acid sequence variants of the anti-MSLN antibodiesdescribed herein are contemplated. For example, in certain embodimentsamino acid sequence variants of anti-MSLN antibodies described hereinare contemplated to improve the binding affinity and/or other biologicalproperties of the antibodies. Exemplary method for preparing amino acidvariants include, but are not limited to, introducing appropriatemodifications into the nucleotide sequence encoding the antibody, or bypeptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of residues within theamino acid sequences of the antibody.

Any combination of deletion, insertion, and substitution can be made toarrive at the final construct, provided that the final constructpossesses the desired characteristics, e.g., antigen-binding. In certainembodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionmutagenesis include the CDRs and framework regions. Examples of suchsubstitutions are described below. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved T-cell mediated cytotoxicity (TDCC). Bothconservative and non-conservative amino acid substitutions arecontemplated for preparing the antibody variants.

In another example of a substitution to create a variant anti-MSLNantibody, one or more hypervariable region residues of a parent antibodyare substituted. In general, variants are then selected based onimprovements in desired properties compared to a parent antibody, forexample, increased affinity, reduced affinity, reduced immunogenicity,increased pH dependence of binding.

In some embodiments, the MSLN binding domain of the MSLN targetingtrispecific protein is a single domain antibody such as a heavy chainvariable domain (VH), a variable domain (VHH) of a llama derived sdAb, apeptide, a ligand or a small molecule entity specific for mesothelin. Insome embodiments, the mesothelin binding domain of the MSLN targetingtrispecific protein described herein is any domain that binds tomesothelin including but not limited to domains from a monoclonalantibody, a polyclonal antibody, a recombinant antibody, a humanantibody, a humanized antibody. In certain embodiments, the MSLN bindingdomain is a single-domain antibody. In other embodiments, the MSLNbinding domain is a peptide. In further embodiments, the MSLN bindingdomain is a small molecule.

Generally, it should be noted that the term single domain antibody asused herein in its broadest sense is not limited to a specificbiological source or to a specific method of preparation. Single domainantibodies are antibodies whose complementary determining regions arepart of a single domain polypeptide. Examples include, but are notlimited to, heavy chain antibodies, antibodies naturally devoid of lightchains, single domain antibodies derived from conventional 4-chainantibodies, engineered antibodies and single domain scaffolds other thanthose derived from antibodies. Single domain antibodies may be any ofthe art, or any future single domain antibodies. Single domainantibodies may be derived from any species including, but not limited tomouse, human, camel, llama, goat, rabbit, bovine. For example, in someembodiments, the single domain antibodies of the disclosure areobtained: (1) by isolating the VHH domain of a naturally occurring heavychain antibody; (2) by expression of a nucleotide sequence encoding anaturally occurring VHH domain; (3) by “humanization” of a naturallyoccurring VHH domain or by expression of a nucleic acid encoding a suchhumanized VHH domain; (4) by “camelization” of a naturally occurring VHdomain from any animal species, and in particular from a species ofmammal, such as from a human being, or by expression of a nucleic acidencoding such a camelized VH domain; (5) by “camelisation” of a “domainantibody” or “Dab”, or by expression of a nucleic acid encoding such acamelized VH domain; (6) by using synthetic or semi-synthetic techniquesfor preparing proteins, polypeptides or other amino acid sequences; (7)by preparing a nucleic acid encoding a single domain antibody usingtechniques for nucleic acid synthesis known in the field, followed byexpression of the nucleic acid thus obtained; and/or (8) by anycombination of one or more of the foregoing.

In one embodiment, a single domain antibody corresponds to the VHHdomains of naturally occurring heavy chain antibodies directed againstMSLN. As further described herein, such VHH sequences can generally begenerated or obtained by suitably immunizing a species of Llama withMSLN, (i.e., so as to raise an immune response and/or heavy chainantibodies directed against MSLN), by obtaining a suitable biologicalsample from said Llama (such as a blood sample, serum sample or sampleof B-cells), and by generating VHH sequences directed against MSLN,starting from said sample, using any suitable technique known in thefield.

In another embodiment, such naturally occurring VHH domains againstMSLN, are obtained from naïve libraries of Camelid VHH sequences, forexample by screening such a library using MSLN, or at least one part,fragment, antigenic determinant or epitope thereof using one or morescreening techniques known in the field. Such libraries and techniquesare for example described in WO 99/37681, WO 01/90190, WO 03/025020 andWO 03/035694. Alternatively, improved synthetic or semi-syntheticlibraries derived from naïve VHH libraries are used, such as VHHlibraries obtained from naïve VHH libraries by techniques such as randommutagenesis and/or CDR shuffling, as for example described in WO00/43507.

In a further embodiment, yet another technique for obtaining VHHsequences directed against MSLN, involves suitably immunizing atransgenic mammal that is capable of expressing heavy chain antibodies(i.e., so as to raise an immune response and/or heavy chain antibodiesdirected against MSLN), obtaining a suitable biological sample from saidtransgenic mammal (such as a blood sample, serum sample or sample ofB-cells), and then generating VHH sequences directed against MSLN,starting from said sample, using any suitable technique known in thefield. For example, for this purpose, the heavy chainantibody-expressing rats or mice and the further methods and techniquesdescribed in WO 02/085945 and in WO 04/049794 can be used.

In some embodiments, an anti-MSLN single domain antibody of the MSLNtargeting trispecific protein comprises a single domain antibody with anamino acid sequence that corresponds to the amino acid sequence of anaturally occurring VHH domain, but that has been “humanized”, i.e., byreplacing one or more amino acid residues in the amino acid sequence ofsaid naturally occurring VHH sequence (and in particular in theframework sequences) by one or more of the amino acid residues thatoccur at the corresponding position(s) in a VH domain from aconventional 4-chain antibody from a human being (e.g., as indicatedabove). This can be performed in a manner known in the field, which willbe clear to the skilled person, for example on the basis of the furtherdescription herein. Again, it should be noted that such humanizedanti-MSLN single domain antibodies of the disclosure are obtained in anysuitable manner known per se (i.e., as indicated under points (1)-(8)above) and thus are not strictly limited to polypeptides that have beenobtained using a polypeptide that comprises a naturally occurring VHHdomain as a starting material. In some additional embodiments, a singledomain anti-MSLN antibody, as described herein, comprises a singledomain antibody with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring VH domain, but that hasbeen “camelized”, i.e., by replacing one or more amino acid residues inthe amino acid sequence of a naturally occurring VH domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a VHH domain of a heavychain antibody. Such “camelizing” substitutions are preferably insertedat amino acid positions that form and/or are present at the VH-VLinterface, and/or at the so-called Camelidae hallmark residues (see forexample WO 94/04678 and Davies and Riechmann (1994 and 1996)).Preferably, the VH sequence that is used as a starting material orstarting point for generating or designing the camelized single domainis preferably a VH sequence from a mammal, more preferably the VHsequence of a human being, such as a VH3 sequence. However, it should benoted that such camelized anti-MSLN single domain antibodies of thedisclosure, in certain embodiments, are obtained in any suitable mannerknown in the field (i.e., as indicated under points (1)-(8) above) andthus are not strictly limited to polypeptides that have been obtainedusing a polypeptide that comprises a naturally occurring VH domain as astarting material. For example, as further described herein, both“humanization” and “camelization” is performed by providing a nucleotidesequence that encodes a naturally occurring VHH domain or VH domain,respectively, and then changing, one or more codons in said nucleotidesequence in such a way that the new nucleotide sequence encodes a“humanized” or “camelized” single domain antibody, respectively. Thisnucleic acid can then be expressed, so as to provide a desired anti-MSLNsingle domain antibody of the disclosure. Alternatively, in otherembodiments, based on the amino acid sequence of a naturally occurringVHH domain or VH domain, respectively, the amino acid sequence of thedesired humanized or camelized anti-MSLN single domain antibody of thedisclosure, respectively, are designed and then synthesized de novousing known techniques for peptide synthesis. In some embodiments, basedon the amino acid sequence or nucleotide sequence of a naturallyoccurring VHH domain or VH domain, respectively, a nucleotide sequenceencoding the desired humanized or camelized anti-MSLN single domainantibody of the disclosure, respectively, is designed and thensynthesized de novo using known techniques for nucleic acid synthesis,after which the nucleic acid thus obtained is expressed in using knownexpression techniques, so as to provide the desired anti-MSLN singledomain antibody of the disclosure.

Other suitable methods and techniques for obtaining the anti-MSLN singledomain antibody of the disclosure and/or nucleic acids encoding thesame, starting from naturally occurring VH sequences or VHH sequencesfor example comprises combining one or more parts of one or morenaturally occurring VH sequences (such as one or more framework (FR)sequences and/or complementarity determining region (CDR) sequences),one or more parts of one or more naturally occurring VHH sequences (suchas one or more FR sequences or CDR sequences), and/or one or moresynthetic or semi-synthetic sequences, in a suitable manner, so as toprovide an anti-MSLN single domain antibody of the disclosure or anucleotide sequence or nucleic acid encoding the same.

In some embodiments, the MSLN binding domain is an anti-MSLN specificantibody comprising a heavy chain variable complementarity determiningregion CDR1, a heavy chain variable CDR2, a heavy chain variable CDR3, alight chain variable CDR1, a light chain variable CDR2, and a lightchain variable CDR3. In some embodiments, the MSLN binding domaincomprises any domain that binds to MSLN including but not limited todomains from a monoclonal antibody, a polyclonal antibody, a recombinantantibody, a human antibody, a humanized antibody, or antigen bindingfragments such as single domain antibodies (sdAb), Fab, Fab′, F(ab)2,and Fv fragments, fragments comprised of one or more CDRs, single-chainantibodies (e.g., single chain Fv fragments (scFv)), disulfidestabilized (dsFv) Fv fragments, heteroconjugate antibodies (e.g.,bispecific antibodies), pFv fragments, heavy chain monomers or dimers,light chain monomers or dimers, and dimers consisting of one heavy chainand one light chain. In some embodiments, the MSLN binding domain is asingle domain antibody. In some embodiments, the anti-MSLN single domainantibody comprises heavy chain variable complementarity determiningregions (CDR), CDR1, CDR2, and CDR3.

In some embodiments, the MSLN binding domain is a polypeptide comprisingan amino acid sequence that is comprised of four frameworkregions/sequences (f1-f4) interrupted by three complementaritydetermining regions/sequences, as represented by the formula:f1-r1-f2-r2-f3-r3-f4, wherein r1, r2, and r3 are complementaritydetermining regions CDR1, CDR2, and CDR3, respectively, and f1, f2, f3,and f4 are framework residues. The framework residues of the MSLNbinding protein of the present disclosure comprise, for example, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or94 amino acid residues, and the complementarity determining regionscomprise, for example, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,or 36 amino acid residues. In some embodiments, the MSLN binding domaincomprises an amino acid sequence selected from SEQ ID NOs: 1-40, and102-105. In some embodiments, the framework region 1 of a MSLN bindingtrispecific protein of this disclosure comprises a sequence as in anyone of SEQ ID Nos.: 223-261. In some embodiments, the framework region 2of a MSLN binding trispecific protein of this disclosure comprises asequence as in any one of SEQ ID Nos.: 262-300. In some embodiments, theframework region 3 of a MSLN binding trispecific protein of thisdisclosure comprises a sequence as in any one of SEQ ID Nos.: 301-339.In some embodiments, the framework region 4 of a MSLN bindingtrispecific protein of this disclosure comprises a sequence as in anyone of SEQ ID Nos.: 340-378.

In some embodiments, the CDR1 comprises the amino acid sequence as setforth in SEQ ID NO: 51 or a variant having one, two, three, four, five,six, seven, eight, nine, or ten amino acid substitutions in SEQ ID NO:51. In some embodiments, the CDR2 comprises a sequence as set forth inSEQ ID NO: 52 or a variant having one, two, three, four, five, six,seven, eight, nine, or ten amino acid substitutions in SEQ ID NO: 52. Insome embodiments, the CDR3 comprises a sequence as set forth in SEQ IDNO: 53 or a variant having one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions in SEQ ID NO: 53.

In some embodiments, the CDR1 comprises the amino acid sequence as setforth in SEQ ID NO: 54 or a variant having one, two, three, four, five,six, seven, eight, nine, or ten amino acid substitutions in SEQ ID NO:54. In some embodiments, the CDR2 comprises a sequence as set forth inSEQ ID NO: 55 or a variant having one, two, three, four, five, six,seven, eight, nine, or ten amino acid substitutions in SEQ ID NO: 55. Insome embodiments, the CDR3 comprises a sequence as set forth in SEQ IDNO: 56 or a variant having one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions in SEQ ID NO: 56.

In some embodiments, the CDR1 comprises the amino acid sequence as setforth in any one of SEQ ID Nos.: 106-144 or a variant having one, two,three, four, five, six, seven, eight, nine, or ten amino acidsubstitutions in any one of SEQ ID Nos.: 106-144. In some embodiments,the CDR2 comprises a sequence as set forth in any one of SEQ ID Nos.:145-183 or a variant having one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions in any one of SEQ ID Nos.:145-183. In some embodiments, the CDR3 comprises a sequence as set forthin any one of SEQ ID Nos.: 184-222 or a variant having one, two, three,four, five, six, seven, eight, nine, or ten amino acid substitutions inany one of SEQ ID Nos.: 184-222.

The MSLN binding domains of the present disclosure, in certain examples,comprise one or more conserved regions. The conserved regions comprisesequences as set forth in SEQ ID NOs: 41-49, or variants comprising oneor more amino acid residue substitutions relative to said sequences.Exemplary embodiments include MSLN binding proteins comprising one ormore conserved regions selected from SEQ ID NOs: 41-44, or variantscomprising one or more amino acid residue substitutions relative to saidsequences. In some cases, the MSLN binding domain comprises (i) astretch of amino acids corresponding to SEQ ID NO: 41, (ii) a stretch ofamino acids corresponding to SEQ ID NO: 42, (iii) a stretch of aminoacids corresponding to SEQ ID NO: 43, and (iv) a stretch of amino acidscorresponding to SEQ ID NO: 44.

Further exemplary embodiments include MSLN binding domains comprisingone or more conserved regions selected from SEQ ID NOs: 45-50, orvariants comprising one or more amino acid residue substitutionsrelative to said sequences. In some cases, the MSLN binding domaincomprises (i) a stretch of amino acids corresponding to SEQ ID NO: 45,(ii) a stretch of amino acids corresponding to SEQ ID NO: 46, (iii) astretch of amino acids corresponding to SEQ ID NO: 47, (iv) a stretch ofamino acids corresponding to SEQ ID NO: 48, (v) a stretch of amino acidcorresponding to SEQ ID NO: 49, and (vi) a stretch of amino acidscorresponding to SEQ ID NO: 50.

In various embodiments, the MSLN binding domain of the presentdisclosure is at least about 75%, about 76%, about 77%, about 78%, about79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or about 100% identical to an amino acid sequence selectedfrom SEQ ID NOs: 1-29.

In various embodiments, the MSLN binding domain of the presentdisclosure is at least about 75%, about 76%, about 77%, about 78%, about79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or about 100% identical to an amino acid sequence selectedfrom SEQ ID NOs: 30-40, and 102-105.

In various embodiments, a complementarity determining region of the MSLNbinding domain of the present disclosure is at least about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100% identical to the amino acid sequence set forth in SEQ ID NO:51, SEQ ID NO: 54, or any one of SEQ ID Nos.: 106-144.

In various embodiments, a complementarity determining region of the MSLNbinding domain of the present disclosure is at least about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100% identical to the amino acid sequence set forth in SEQ ID NO:52, SEQ ID NO: 55, or any one of SEQ ID Nos.: 145-183.

In various embodiments, a complementarity determining region of the MSLNbinding domain of the present disclosure is at least about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100% identical to the amino acid sequence set forth in SEQ ID NO:53, SEQ ID NO: 56, or any one of SEQ ID Nos.: 184-222.

In some embodiments, the MSLN binding protein, according to any one ofthe above embodiments, is a single domain antibody comprising thesequence of SEQ ID NO: 1. In some embodiments, the MSLN binding protein,according to any one of the above embodiments, is a single domainantibody comprising the sequence of SEQ ID NO: 2. In some embodiments,the MSLN binding protein, according to any one of the above embodiments,is a single domain antibody comprising the sequence of SEQ ID NO: 3. Insome embodiments, the MSLN binding protein, according to any one of theabove embodiments, is a single domain antibody comprising the sequenceof SEQ ID NO: 4. In some embodiments, the MSLN binding protein,according to any one of the above embodiments, is a single domainantibody comprising the sequence of SEQ ID NO: 5. In some embodiments,the MSLN binding protein, according to any one of the above embodiments,is a single domain antibody comprising the sequence of SEQ ID NO: 6. Insome embodiments, the MSLN binding protein, according to any one of theabove embodiments, is a single domain antibody comprising the sequenceof SEQ ID NO: 7. In some embodiments, the MSLN binding protein,according to any one of the above embodiments, is a single domainantibody comprising the sequence of SEQ ID NO: 8. In some embodiments,the MSLN binding protein, according to any one of the above embodiments,is a single domain antibody comprising the sequence of SEQ ID NO: 9. Insome embodiments, the MSLN binding protein, according to any one of theabove embodiments, is a single domain antibody comprising the sequenceof SEQ ID NO: 10. In some embodiments, the MSLN binding protein,according to any one of the above embodiments, is a single domainantibody comprising the sequence of SEQ ID NO: 11. In some embodiments,the MSLN binding protein, according to any one of the above embodiments,is a single domain antibody comprising the sequence of SEQ ID NO: 12. Insome embodiments, the MSLN binding protein, according to any one of theabove embodiments, is a single domain antibody comprising the sequenceof SEQ ID NO: 13. In some embodiments, the MSLN binding protein,according to any one of the above embodiments, is a single domainantibody comprising the sequence of SEQ ID NO: 14. In some embodiments,the MSLN binding protein, according to any one of the above embodiments,is a single domain antibody comprising the sequence of SEQ ID NO: 15. Insome embodiments, the MSLN binding protein, according to any one of theabove embodiments, is a single domain antibody comprising the sequenceof SEQ ID NO: 16. In some embodiments, the MSLN binding protein,according to any one of the above embodiments, is a single domainantibody comprising the sequence of SEQ ID NO: 17. In some embodiments,the MSLN binding protein, according to any one of the above embodiments,is a single domain antibody comprising the sequence of SEQ ID NO: 18. Insome embodiments, the MSLN binding protein, according to any one of theabove embodiments, is a single domain antibody comprising the sequenceof SEQ ID NO: 19. In some embodiments, the MSLN binding protein,according to any one of the above embodiments, is a single domainantibody comprising the sequence of SEQ ID NO: 20. In some embodiments,the MSLN binding protein, according to any one of the above embodiments,is a single domain antibody comprising the sequence of SEQ ID NO: 21. Insome embodiments, the MSLN binding protein, according to any one of theabove embodiments, is a single domain antibody comprising the sequenceof SEQ ID NO: 22. In some embodiments, the MSLN binding protein,according to any one of the above embodiments, is a single domainantibody comprising the sequence of SEQ ID NO: 23. In some embodiments,the MSLN binding protein, according to any one of the above embodiments,is a single domain antibody comprising the sequence of SEQ ID NO: 24. Insome embodiments, the MSLN binding protein, according to any one of theabove embodiments, is a single domain antibody comprising the sequenceof SEQ ID NO: 25. In some embodiments, the MSLN binding protein,according to any one of the above embodiments, is a single domainantibody comprising the sequence of SEQ ID NO: 26. In some embodiments,the MSLN binding protein, according to any one of the above embodiments,is a single domain antibody comprising the sequence of SEQ ID NO: 27. Insome embodiments, the MSLN binding protein, according to any one of theabove embodiments, is a single domain antibody comprising the sequenceof SEQ ID NO: 28. In some embodiments, the MSLN binding protein,according to any one of the above embodiments, is a single domainantibody comprising the sequence of SEQ ID NO: 29.

In some embodiments, the MSLN binding protein, according to any one ofthe above embodiments, is a humanized single domain antibody comprisingthe sequence of SEQ ID NO: 30. In some embodiments, the MSLN bindingprotein, according to any one of the above embodiments, is a humanizedsingle domain antibody comprising the sequence of SEQ ID NO: 31. In someembodiments, the MSLN binding protein, according to any one of the aboveembodiments, is a humanized single domain antibody comprising thesequence of SEQ ID NO: 32. In some embodiments, the MSLN bindingprotein, according to any one of the above embodiments, is a humanizedsingle domain antibody comprising the sequence of SEQ ID NO: 33. In someembodiments, the MSLN binding protein, according to any one of the aboveembodiments, is a humanized single domain antibody comprising thesequence of SEQ ID NO: 34. In some embodiments, the MSLN bindingprotein, according to any one of the above embodiments, is a humanizedsingle domain antibody comprising the sequence of SEQ ID NO: 35. In someembodiments, the MSLN binding protein, according to any one of the aboveembodiments, is a humanized single domain antibody comprising thesequence of SEQ ID NO: 36. In some embodiments, the MSLN bindingprotein, according to any one of the above embodiments, is a humanizedsingle domain antibody comprising the sequence of SEQ ID NO: 37. In someembodiments, the MSLN binding protein, according to any one of the aboveembodiments, is a humanized single domain antibody comprising thesequence of SEQ ID NO: 38. In some embodiments, the MSLN bindingprotein, according to any one of the above embodiments, is a humanizedsingle domain antibody comprising the sequence of SEQ ID NO: 39. In someembodiments, the MSLN binding protein, according to any one of the aboveembodiments, is a humanized single domain antibody comprising thesequence of SEQ ID NO: 40. In some embodiments, the MSLN bindingprotein, according to any one of the above embodiments, is a humanizedsingle domain antibody comprising the sequence of SEQ ID NO: 102. Insome embodiments, the MSLN binding protein, according to any one of theabove embodiments, is a humanized single domain antibody comprising thesequence of SEQ ID NO: 103. In some embodiments, the MSLN bindingprotein, according to any one of the above embodiments, is a humanizedsingle domain antibody comprising the sequence of SEQ ID NO: 104. Insome embodiments, the MSLN binding protein, according to any one of theabove embodiments, is a humanized single domain antibody comprising thesequence of SEQ ID NO: 105.

In some embodiments, the MSLN binding domain is cross-reactive withhuman and cynomolgus mesothelin. In some embodiments, the MSLN bindingdomain is specific for human mesothelin. In certain embodiments, theMSLN binding domains disclosed herein bind to human mesothelin with ahuman Kd (hKd). In certain embodiments, the MSLN binding domainsdisclosed herein bind to cynomolgus mesothelin with a cyno Kd (cKd). Incertain embodiments, the MSLN binding domains disclosed herein bind toboth cynomolgus mesothelin and a human mesothelin, with a cyno Kd (cKd)and a human Kd (hKd), respectively. In some embodiments, the MSLNbinding protein binds to human and cynomolgus mesothelin with comparablebinding affinities (i.e., hKd and cKd values do not differ by more than±10%). In some embodiments, the hKd and the cKd range from about 0.1 nMto about 500 nM. In some embodiments, the hKd and the cKd range fromabout 0.1 nM to about 450 nM. In some embodiments, the hKd and the cKdrange from about 0.1 nM to about 400 nM. In some embodiments, the hKdand the cKd range from about 0.1 nM to about 350 nM. In someembodiments, the hKd and the cKd range from about 0.1 nM to about 300nM. In some embodiments, the hKd and the cKd range from about 0.1 nM toabout 250 nM. In some embodiments, the hKd and the cKd range from about0.1 nM to about 200 nM. In some embodiments, the hKd and the cKd rangefrom about 0.1 nM to about 150 nM. In some embodiments, the hKd and thecKd range from about 0.1 nM to about 100 nM. In some embodiments, thehKd and the cKd range from about 0.1 nM to about 90 nM. In someembodiments, the hKd and the cKd range from about 0.2 nM to about 80 nM.In some embodiments, the hKd and the cKd range from about 0.3 nM toabout 70 nM. In some embodiments, the hKd and the cKd range from about0.4 nM to about 50 nM. In some embodiments, the hKd and the cKd rangefrom about 0.5 nM to about 30 nM. In some embodiments, the hKd and thecKd range from about 0.6 nM to about 10 nM. In some embodiments, the hKdand the cKd range from about 0.7 nM to about 8 nM. In some embodiments,the hKd and the cKd range from about 0.8 nM to about 6 nM. In someembodiments, the hKd and the cKd range from about 0.9 nM to about 4 nM.In some embodiments, the hKd and the cKd range from about 1 nM to about2 nM.

In some embodiments, any of the foregoing MSLN binding domains (e.g.,anti-MSLN single domain antibodies of SEQ ID NOs: 1-40) are affinitypeptide tagged for ease of purification. In some embodiments, theaffinity peptide tag is six consecutive histidine residues, alsoreferred to as 6×-his (SEQ ID NO: 379).

In certain embodiments, the MSLN binding domains of the presentdisclosure preferentially bind membrane bound mesothelin over solublemesothelin. Membrane bound mesothelin refers to the presence ofmesothelin in or on the cell membrane surface of a cell that expressesmesothelin. Soluble mesothelin refers to mesothelin that is no longer onin or on the cell membrane surface of a cell that expresses or expressedmesothelin. In certain instances, the soluble mesothelin is present inthe blood and/or lymphatic circulation in a subject. In one embodiment,the MSLN binding domains bind membrane-bound mesothelin at least 5 fold,10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 40 fold, 50 fold, 100 fold,500 fold, or 1000 fold greater than soluble mesothelin. In oneembodiment, the MSLN targeting trispecific antigen binding proteins ofthe present disclosure preferentially bind membrane-bound mesothelin 30fold greater than soluble mesothelin. Determining the preferentialbinding of an antigen binding protein to membrane bound MSLN oversoluble MSLN can be readily determined using assays well known in theart.

TriTAC Molecules

Various embodiments of this disclosure provides a trispecific molecule(also referred to herein as a TriTAC molecule) comprising a MSLN bindingdomain as described herein. In some embodiments, the TriTAC moleculecomprises an amino acid sequence as set forth in any one of SEQ ID Nos:58-86, 98, 100, and 101. In some embodiments, a TriTAC molecule of thisdisclosure comprises an amino acid sequence that is at least about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100% identicalto an amino acid sequence selected from SEQ ID NOs: 58-86, 98, 100, and101. In some embodiments, a TriTAC molecule of this disclosure comprisesan amino acid sequence that is at least about 75%, about 76%, about 77%,about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, or about 100% identical to the full length ofan amino acid sequence selected from SEQ ID NOs: 58-86, 98, 100, and101. In some embodiments, a TriTAC molecule of this disclosure comprisesan amino acid sequence that is at least about 75%, about 76%, about 77%,about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, or about 100% identical to a fraction of thefull length of an amino acid sequence selected from SEQ ID NOs: 58-86,98, 100, and 101.

Integration into Chimeric Antigen Receptors (CAR)

The MSLN targeting trispecific antigen binding proteins of the presentdisclosure can, in certain examples, be incorporated into a chimericantigen receptor (CAR). An engineered immune effector cell, e.g., a Tcell or NK cell, can be used to express a CAR that includes an anti-MSLNtargeting trispecific protein containing an anti-MSLN single domainantibody as described herein. In one embodiment, the CAR including ananti-MSLN targeting trispecific protein as described herein is connectedto a transmembrane domain via a hinge region, and further acostimulatory domain, e.g., a functional signaling domain obtained fromOX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), or4-1BB. In some embodiments, the CAR further comprises a sequenceencoding a intracellular signaling domain, such as 4-1BB and/or CD3zeta.

Tumor Growth Reduction Properties

In certain embodiments, the MSLN targeting trispecific proteins of thedisclosure reduce the growth of tumor cells in vivo when administered toa subject who has tumor cells that express mesothelin. Measurement ofthe reduction of the growth of tumor cells can be determined by multipledifferent methodologies well known in the art. Nonlimiting examplesinclude direct measurement of tumor dimension, measurement of excisedtumor mass and comparison to control subjects, measurement via imagingtechniques (e.g., CT or MRI) that may or may not use isotopes orluminescent molecules (e.g., luciferase) for enhanced analysis, and thelike. In specific embodiments, administration of the trispecificproteins of the disclosure results in a reduction of in vivo growth oftumor cells as compared to a control antigen binding agent by at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, with an about100% reduction in tumor growth indicating a complete response anddisappearance of the tumor. In further embodiments, administration ofthe trispecific proteins of the disclosure results in a reduction of invivo growth of tumor cells as compared to a control antigen bindingagent by about 50-100%, about 75-100% or about 90-100%. In furtherembodiments, administration of the trispecific proteins of thedisclosure results in a reduction of in vivo growth of tumor cells ascompared to a control antigen binding agent by about 50-60%, about60-70%, about 70-80%, about 80-90%, or about 90-100%.

MSLN Trispecific Protein Modifications

The MSLN targeting trispecific proteins described herein encompassderivatives or analogs in which (i) an amino acid is substituted with anamino acid residue that is not one encoded by the genetic code, (ii) themature polypeptide is fused with another compound such as polyethyleneglycol, or (iii) additional amino acids are fused to the protein, suchas a leader or secretory sequence or a sequence for purification of theprotein.

Typical modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphatidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Modifications are made anywhere in MSLN targeting trispecific proteinsdescribed herein, including the peptide backbone, the amino acidside-chains, and the amino or carboxyl termini. Certain common peptidemodifications that are useful for modification of MSLN targetingtrispecific proteins include glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation, blockageof the amino or carboxyl group in a polypeptide, or both, by a covalentmodification, and ADP-ribosylation.

Polynucleotides Encoding MSLN Targeting Trispecific Proteins

Also provided, in some embodiments, are polynucleotide moleculesencoding an anti-MSLN trispecific binding protein described herein. Insome embodiments, the polynucleotide molecules are provided as a DNAconstruct. In other embodiments, the polynucleotide molecules areprovided as a messenger RNA transcript.

The polynucleotide molecules are constructed by known methods such as bycombining the genes encoding the three binding domains either separatedby peptide linkers or, in other embodiments, directly linked by apeptide bond, into a single genetic construct operably linked to asuitable promoter, and optionally a suitable transcription terminator,and expressing it in bacteria or other appropriate expression systemsuch as, for example CHO cells. In the embodiments where the MSLNbinding domain is a small molecule, the polynucleotides contain genesencoding the CD3 binding domain and the half-life extension domain. Inthe embodiments where the half-life extension domain is a smallmolecule, the polynucleotides contain genes encoding the domains thatbind to CD3 and MSLN. Depending on the vector system and host utilized,any number of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. The promoter isselected such that it drives the expression of the polynucleotide in therespective host cell.

In some embodiments, the polynucleotide is inserted into a vector,preferably an expression vector, which represents a further embodiment.This recombinant vector can be constructed according to known methods.Vectors of particular interest include plasmids, phagemids, phagederivatives, virii (e.g., retroviruses, adenoviruses, adeno-associatedviruses, herpes viruses, lentiviruses, and the like), and cosmids.

A variety of expression vector/host systems may be utilized to containand express the polynucleotide encoding the polypeptide of the describedtrispecific antigen-binding protein. Examples of expression vectors forexpression in E. coli are pSKK (Le Gall et al., J Immunol Methods.(2004) 285(1): 111-27) or pcDNA5 (Invitrogen) for expression inmammalian cells.

Thus, the MSLN targeting trispecific proteins as described herein, insome embodiments, are produced by introducing a vector encoding theprotein as described above into a host cell and culturing said host cellunder conditions whereby the protein domains are expressed, may beisolated and, optionally, further purified.

Pharmaceutical Compositions

Also provided, in some embodiments, are pharmaceutical compositionscomprising an anti-MSLN trispecific binding protein described herein, avector comprising the polynucleotide encoding the polypeptide of theMSLN targeting trispecific proteins or a host cell transformed by thisvector and at least one pharmaceutically acceptable carrier. The term“pharmaceutically acceptable carrier” includes, but is not limited to,any carrier that does not interfere with the effectiveness of thebiological activity of the ingredients and that is not toxic to thepatient to whom it is administered. Examples of suitable pharmaceuticalcarriers are well known in the art and include phosphate buffered salinesolutions, water, emulsions, such as oil/water emulsions, various typesof wetting agents, sterile solutions etc. Such carriers can beformulated by conventional methods and can be administered to thesubject at a suitable dose. Preferably, the compositions are sterile.These compositions may also contain adjuvants such as preservative,emulsifying agents and dispersing agents. Prevention of the action ofmicroorganisms may be ensured by the inclusion of various antibacterialand antifungal agents. A further embodiment provides one or more of theabove described MSLN targeting trispecific proteins packaged inlyophilized form, or packaged in an aqueous medium.

In some embodiments of the pharmaceutical compositions, the MSLNtargeting trispecific proteins described herein are encapsulated innanoparticles. In some embodiments, the nanoparticles are fullerenes,liquid crystals, liposome, quantum dots, superparamagneticnanoparticles, dendrimers, or nanorods. In other embodiments of thepharmaceutical compositions, the MSLN trispecific antigen-bindingprotein is attached to liposomes. In some instances, the MSLNtrispecific antigen-binding protein are conjugated to the surface ofliposomes. In some instances, the MSLN trispecific antigen-bindingprotein are encapsulated within the shell of a liposome. In someinstances, the liposome is a cationic liposome.

The MSLN targeting trispecific proteins described herein arecontemplated for use as a medicament. Administration is effected bydifferent ways, e.g. by intravenous, intraperitoneal, subcutaneous,intramuscular, topical or intradermal administration. In someembodiments, the route of administration depends on the kind of therapyand the kind of compound contained in the pharmaceutical composition.The dosage regimen will be determined by the attending physician andother clinical factors. Dosages for any one patient depends on manyfactors, including the patient's size, body surface area, age, sex, theparticular compound to be administered, time and route ofadministration, the kind of therapy, general health and other drugsbeing administered concurrently. An “effective dose” refers to amountsof the active ingredient that are sufficient to affect the course andthe severity of the disease, leading to the reduction or remission ofsuch pathology and may be determined using known methods.

In some embodiments, the MSLN targeting trispecific proteins of thisdisclosure are administered at a dosage of up to 10 mg/kg at a frequencyof once a week. In some cases, the dosage ranges from about 1 ng/kg toabout 10 mg/kg. In some embodiments, the dose is from about 1 ng/kg toabout 10 ng/kg, about 5 ng/kg to about 15 ng/kg, about 12 ng/kg to about20 ng/kg, about 18 ng/kg to about 30 ng/kg, about 25 ng/kg to about 50ng/kg, about 35 ng/kg to about 60 ng/kg, about 45 ng/kg to about 70ng/kg, about 65 ng/kg to about 85 ng/kg, about 80 ng/kg to about 1μg/kg, about 0.5 μg/kg to about 5 μg/kg, about 2 μg/kg to about 10μg/kg, about 7 μg/kg to about 15 μg/kg, about 12 μg/kg to about 25μg/kg, about 20 μg/kg to about 50 μg/kg, about 35 μg/kg to about 70μg/kg, about 45 μg/kg to about 80 μg/kg, about 65 μg/kg to about 90μg/kg, about 85 μg/kg to about 0.1 mg/kg, about 0.095 mg/kg to about 10mg/kg. In some cases, the dosage is about 0.1 mg/kg to about 0.2 mg/kg;about 0.25 mg/kg to about 0.5 mg/kg, about 0.45 mg/kg to about 1 mg/kg,about 0.75 mg/kg to about 3 mg/kg, about 2.5 mg/kg to about 4 mg/kg,about 3.5 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 6 mg/kg,about 5.5 mg/kg to about 7 mg/kg, about 6.5 mg/kg to about 8 mg/kg,about 7.5 mg/kg to about 9 mg/kg, or about 8.5 mg/kg to about 10 mg/kg.The frequency of administration, in some embodiments, is about less thandaily, every other day, less than once a day, twice a week, weekly, oncein 7 days, once in two weeks, once in two weeks, once in three weeks,once in four weeks, or once a month. In some cases, the frequency ofadministration is weekly. In some cases, the frequency of administrationis weekly and the dosage is up to 10 mg/kg. In some cases, duration ofadministration is from about 1 day to about 4 weeks or longer.

Methods of Treatment

Also provided herein, in some embodiments, are methods and uses forstimulating the immune system of an individual in need thereofcomprising administration of an anti-MSLN targeting trispecific proteinas described herein. In some instances, the administration of ananti-MSLN targeting trispecific protein described herein induces and/orsustains cytotoxicity towards a cell expressing a target antigen. Insome instances, the cell expressing a target antigen is a cancer ortumor cell, a virally infected cell, a bacterially infected cell, anautoreactive T or B cell, damaged red blood cells, arterial plaques, orfibrotic tissue.

Also provided herein are methods and uses for a treatment of a disease,disorder or condition associated with a target antigen comprisingadministering to an individual in need thereof an anti-MSLN targetingtrispecific protein described herein. Diseases, disorders or conditionsassociated with a target antigen include, but are not limited to, viralinfection, bacterial infection, auto-immune disease, transplantrejection, atherosclerosis, or fibrosis. In other embodiments, thedisease, disorder or condition associated with a target antigen is aproliferative disease, a tumorous disease, an inflammatory disease, animmunological disorder, an autoimmune disease, an infectious disease, aviral disease, an allergic reaction, a parasitic reaction, agraft-versus-host disease or a host-versus-graft disease. In oneembodiment, the disease, disorder or condition associated with a targetantigen is cancer. Cancers that can be treated, prevented, or managed bythe MSLN binding proteins of the present disclosure, and methods ofusing them, include but are not limited to cancers of an epithelial cellorigin. Examples of such cancers include the following: leukemias, suchas but not limited to, acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemias, such as, myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia leukemias andmyelodysplastic syndrome; chronic leukemias, such as but not limited to,chronic myelocytic (granulocytic) leukemia, chronic lymphocyticleukemia, hairy cell leukemia; polycythemia vera; lymphomas such as butnot limited to Hodgkin's disease, non-Hodgkin's disease; multiplemyelomas such as but not limited to smoldering multiple myeloma,nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia,solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom'smacroglobulinemia; monoclonal gammopathy of undetermined significance;benign monoclonal gammopathy; heavy chain disease; bone and connectivetissue sarcomas such as but not limited to bone sarcoma, osteosarcoma,chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor,fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissuesarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi'ssarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma,rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limitedto, glioma, astrocytoma, brain stem glioma, ependymoma,oligodendroglioma, nonglial tumor, acoustic neurinoma,craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including but notlimited to ductal carcinoma, adenocarcinoma, lobular (small cell)carcinoma, intraductal carcinoma, medullary breast cancer, mucinousbreast cancer, tubular breast cancer, papillary breast cancer, Paget'sdisease, and inflammatory breast cancer; adrenal cancer such as but notlimited to pheochromocytom and adrenocortical carcinoma; thyroid cancersuch as but not limited to papillary or follicular thyroid cancer,medullary thyroid cancer and anaplastic thyroid cancer; pancreaticcancer such as but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers such as but limited to Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers such as but not limited to ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers such as squamous cell carcinoma,adenocarcinoma, and melanoma; vulvar cancer such as squamous cellcarcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, andPaget's disease; cervical cancers such as but not limited to, squamouscell carcinoma, and adenocarcinoma; uterine cancers such as but notlimited to endometrial carcinoma and uterine sarcoma; ovarian cancerssuch as but not limited to, ovarian epithelial carcinoma, borderlinetumor, germ cell tumor, and stromal tumor; esophageal cancers such asbut not limited to, squamous cancer, adenocarcinoma, adenoid cysticcarcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell)carcinoma; stomach cancers such as but not limited to, adenocarcinoma,fungating (polypoid), ulcerating, superficial spreading, diffuselyspreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma; colon cancers; rectal cancers; liver cancers such as butnot limited to hepatocellular carcinoma and hepatoblastoma; gallbladdercancers such as adenocarcinoma; cholangiocarcinomas such as but notlimited to pappillary, nodular, and diffuse; lung cancers such asnon-small cell lung cancer, squamous cell carcinoma (epidermoidcarcinoma), adenocarcinoma, large-cell carcinoma and small-cell lungcancer; testicular cancers such as but not limited to germinal tumor,seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma,embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sactumor), prostate cancers such as but not limited to, prostaticintraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers such as but not limited tosquamous cell carcinoma; basal cancers; salivary gland cancers such asbut not limited to adenocarcinoma, mucoepidermoid carcinoma, andadenoidcystic carcinoma; pharynx cancers such as but not limited tosquamous cell cancer, and verrucous; skin cancers such as but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers such as but notlimited to renal cell carcinoma, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers such as but not limited to transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendothelio sarcoma, mesothelioma,synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,bronchogenic carcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma and papillary adenocarcinomas (for areview of such disorders, see Fishman et al., 1985, Medicine, 2d Ed.,J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, InformedDecisions: The Complete Book of Cancer Diagnosis, Treatment, andRecovery, Viking Penguin, Penguin Books U.S.A., Inc., United States ofAmerica).

The MSLN targeting trispecific proteins of the disclosure are alsouseful in the treatment or prevention of a variety of cancers or otherabnormal proliferative diseases, including (but not limited to) thefollowing: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;including squamous cell carcinoma; hematopoietic tumors of lymphoidlineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt'slymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including melanoma, seminoma, tetratocarcinoma, neuroblastomaand glioma; tumors of the central and peripheral nervous system,including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyoscarama, andosteosarcoma; and other tumors, including melanoma, xerodermapigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma. It is also contemplated that cancers caused byaberrations in apoptosis would also be treated by the methods andcompositions of the disclosure. Such cancers may include but not belimited to follicular lymphomas, carcinomas with p53 mutations, hormonedependent tumors of the breast, prostate and ovary, and precancerouslesions such as familial adenomatous polyposis, and myelodysplasticsyndromes. In specific embodiments, malignancy or dysproliferativechanges (such as metaplasias and dysplasias), or hyperproliferativedisorders, are treated or prevented in the skin, lung, colon, breast,prostate, bladder, kidney, pancreas, ovary, or uterus. In other specificembodiments, sarcoma, melanoma, or leukemia is treated or prevented.

As used herein, in some embodiments, “treatment” or “treating” or“treated” refers to therapeutic treatment wherein the object is to slow(lessen) an undesired physiological condition, disorder or disease, orto obtain beneficial or desired clinical results. For the purposesdescribed herein, beneficial or desired clinical results include, butare not limited to, alleviation of symptoms; diminishment of the extentof the condition, disorder or disease; stabilization (i.e., notworsening) of the state of the condition, disorder or disease; delay inonset or slowing of the progression of the condition, disorder ordisease; amelioration of the condition, disorder or disease state; andremission (whether partial or total), whether detectable orundetectable, or enhancement or improvement of the condition, disorderor disease. Treatment includes eliciting a clinically significantresponse without excessive levels of side effects. Treatment alsoincludes prolonging survival as compared to expected survival if notreceiving treatment. In other embodiments, “treatment” or “treating” or“treated” refers to prophylactic measures, wherein the object is todelay onset of or reduce severity of an undesired physiologicalcondition, disorder or disease, such as, for example is a person who ispredisposed to a disease (e.g., an individual who carries a geneticmarker for a disease such as breast cancer).

In some embodiments of the methods described herein, the MSLN targetingtrispecific proteins as described herein are administered in combinationwith an agent for treatment of the particular disease, disorder orcondition. Agents include but are not limited to, therapies involvingantibodies, small molecules (e.g., chemotherapeutics), hormones(steroidal, peptide, and the like), radiotherapies (γ-rays, X-rays,and/or the directed delivery of radioisotopes, microwaves, UV radiationand the like), gene therapies (e.g., antisense, retroviral therapy andthe like) and other immunotherapies. In some embodiments, an anti-MSLNtargeting trispecific protein as described herein is administered incombination with anti-diarrheal agents, anti-emetic agents, analgesics,opioids and/or non-steroidal anti-inflammatory agents. In someembodiments, an anti-MSLN targeting trispecific protein as describedherein is administered in combination with anti-cancer agents.Nonlimiting examples of anti-cancer agents that can be used in thevarious embodiments of the disclosure, including pharmaceuticalcompositions and dosage forms and kits of the disclosure, include:acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin;aldesleukin; altretamine; ambomycin; ametantrone acetate;aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicinhydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin;enpromate; epipropidine; epirubicin hydrochloride; erbulozole;esorubicin hydrochloride; estramustine; estramustine phosphate sodium;etanidazole; etoposide; etoposide phosphate; etoprine; fadrozolehydrochloride; fazarabine; fenretinide; floxuridine; fludarabinephosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; ilmofosine; interleukin II (includingrecombinant interleukin II, or rIL2), interferon alpha-2a; interferonalpha-2b; interferon alpha-n1 interferon alpha-n3; interferon beta-I a;interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotideacetate; letrozole; leuprolide acetate; liarozole hydrochloride;lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol;maytansine; mechlorethamine hydrochloride; megestrol acetate;melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide;mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper;mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole;nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium;tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinzolidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride. Other examples of anti-cancer drugs include, but are notlimited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil;abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin;aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox;amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide;anastrozole; andrographolide; angiogenesis inhibitors; antagonist D;antagonist G; antarelix; anti-dorsalizing morphogenetic protein-i;antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;antisense oligonucleotides; aphidicolin glycinate; apoptosis genemodulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenylspiromustine; docetaxel; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin;epristeride; estramustine analogue; estrogen agonists; estrogenantagonists; etanidazole; etoposide phosphate; exemestane; fadrozole;fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-I receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; HMG-CoA reductase inhibitor (suchas but not limited to, Lovastatin, Pravastatin, Fluvastatin, Statin,Simvastatin, and Atorvastatin); loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;marimastat; masoprocol; maspin; matrilysin inhibitors; matrixmetalloproteinase inhibitors; menogaril; merbarone; meterelin;methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine;mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol;mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;Vitaxin®; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Additional anti-cancer drugs are 5-fluorouracil andleucovorin. These two agents are particularly useful when used inmethods employing thalidomide and a topoisomerase inhibitor. In someembodiments, the anti-MSLN targeting trispecific protein of the presentdisclosure is used in combination with gemcitabine.

In some embodiments, the anti-MSLN targeting trispecific protein asdescribed herein is administered before, during, or after surgery.

Methods of Detection of Mesothelin Expression and Diagnosis ofMesothelin Associated Cancer

According to another embodiment of the disclosure, kits for detectingexpression of mesothelin in vitro or in vivo are provided. The kitsinclude the foregoing MSLN targeting trispecific proteins (e.g., atrispecific protein containing a labeled anti-MSLN single domainantibody or antigen binding fragments thereof), and one or morecompounds for detecting the label. In some embodiments, the label isselected from the group consisting of a fluorescent label, an enzymelabel, a radioactive label, a nuclear magnetic resonance active label, aluminescent label, and a chromophore label.

In some cases, mesothelin expression is detected in a biological sample.The sample can be any sample, including, but not limited to, tissue frombiopsies, autopsies and pathology specimens. Biological samples alsoinclude sections of tissues, for example, frozen sections taken forhistological purposes. Biological samples further include body fluids,such as blood, serum, plasma, sputum, spinal fluid or urine. Abiological sample is typically obtained from a mammal, such as a humanor non-human primate.

In one embodiment, provided is a method of determining if a subject hascancer by contacting a sample from the subject with an anti-MSLN singledomain antibody as disclosed herein; and detecting binding of the singledomain antibody to the sample. An increase in binding of the antibody tothe sample as compared to binding of the antibody to a control sampleidentifies the subject as having cancer.

In another embodiment, provided is a method of confirming a diagnosis ofcancer in a subject by contacting a sample from a subject diagnosed withcancer with an anti-MSLN single domain antibody as disclosed herein; anddetecting binding of the antibody to the sample. An increase in bindingof the antibody to the sample as compared to binding of the antibody toa control sample confirms the diagnosis of cancer in the subject.

In some examples of the disclosed methods, the MSLN single domainantibody of the trispecific protein is directly labeled.

In some examples, the methods further include contacting a secondantibody that specifically binds the anti-MSLN single domain antibodywith the sample; and detecting the binding of the second antibody. Anincrease in binding of the second antibody to the sample as compared tobinding of the second antibody to a control sample detects cancer in thesubject or confirms the diagnosis of cancer in the subject.

In some cases, the cancer is mesothelioma, prostate cancer, lung cancer,stomach cancer, squamous cell carcinoma, pancreatic cancer,cholangiocarcinoma, triple negative breast cancer or ovarian cancer, orany other type of cancer that expresses mesothelin.

In some examples, the control sample is a sample from a subject withoutcancer. In particular examples, the sample is a blood or tissue sample.

In some cases, the antibody that binds (for example specifically binds)mesothelin is directly labeled with a detectable label. In anotherembodiment, the antibody that binds (for example, specifically binds)mesothelin (the first antibody) is unlabeled and a second antibody orother molecule that can bind the antibody that specifically bindsmesothelin is labeled. A second antibody is chosen such that it is ableto specifically bind the specific species and class of the firstantibody. For example, if the first antibody is a llama IgG, then thesecondary antibody may be an anti-llama-IgG. Other molecules that canbind to antibodies include, without limitation, Protein A and Protein G,both of which are available commercially. Suitable labels for theantibody or secondary antibody are described above, and include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, magnetic agents and radioactive materials. Non-limitingexamples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase. Nonlimitingexamples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin. Nonlimiting examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin. A non-limiting exemplary luminescent materialis luminol; a non-limiting exemplary a magnetic agent is gadolinium, andnon-limiting exemplary radioactive labels include 125I, 131I, 35S or 3H.

In an alternative embodiment, mesothelin can be assayed in a biologicalsample by a competition immunoassay utilizing mesothelin standardslabeled with a detectable substance and an unlabeled antibody thatspecifically binds mesothelin. In this assay, the biological sample, thelabeled mesothelin standards and the antibody that specifically bindmesothelin are combined and the amount of labeled mesothelin standardbound to the unlabeled antibody is determined. The amount of mesothelinin the biological sample is inversely proportional to the amount oflabeled mesothelin standard bound to the antibody that specificallybinds mesothelin.

The immunoassays and method disclosed herein can be used for a number ofpurposes. In one embodiment, the antibody that specifically bindsmesothelin may be used to detect the production of mesothelin in cellsin cell culture. In another embodiment, the antibody can be used todetect the amount of mesothelin in a biological sample, such as a tissuesample, or a blood or serum sample. In some examples, the mesothelin iscell-surface mesothelin. In other examples, the mesothelin is solublemesothelin (e.g., mesothelin in a cell culture supernatant or solublemesothelin in a body fluid sample, such as a blood or serum sample).

In one embodiment, a kit is provided for detecting mesothelin in abiological sample, such as a blood sample or tissue sample. For example,to confirm a cancer diagnosis in a subject, a biopsy can be performed toobtain a tissue sample for histological examination. Alternatively, ablood sample can be obtained to detect the presence of solublemesothelin protein or fragment. Kits for detecting a polypeptide willtypically comprise a single domain antibody, according to the presentdisclosure, that specifically binds mesothelin. In some embodiments, anantibody fragment, such as an scFv fragment, a VH domain, or a Fab isincluded in the kit. In a further embodiment, the antibody is labeled(for example, with a fluorescent, radioactive, or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use of an antibody that binds mesothelin. The instructionalmaterials may be written, in an electronic form (such as a computerdiskette or compact disk) or may be visual (such as video files). Thekits may also include additional components to facilitate the particularapplication for which the kit is designed. Thus, for example, the kitmay additionally contain means of detecting a label (such as enzymesubstrates for enzymatic labels, filter sets to detect fluorescentlabels, appropriate secondary labels such as a secondary antibody, orthe like). The kits may additionally include buffers and other reagentsroutinely used for the practice of a particular method. Such kits andappropriate contents are well known to those of skill in the art.

In one embodiment, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting mesothelin in a biological samplegenerally includes the steps of contacting the biological sample with anantibody which specifically reacts, under immunologically reactiveconditions, to a mesothelin polypeptide. The antibody is allowed tospecifically bind under immunologically reactive conditions to form animmune complex, and the presence of the immune complex (bound antibody)is detected directly or indirectly.

Methods of determining the presence or absence of a cell surface markerare well known in the art. For example, the antibodies can be conjugatedto other compounds including, but not limited to, enzymes, magneticbeads, colloidal magnetic beads, haptens, fluorochromes, metalcompounds, radioactive compounds or drugs. The antibodies can also beutilized in immunoassays such as but not limited to radioimmunoassays(RIAs), ELISA, or immunohistochemical assays. The antibodies can also beused for fluorescence activated cell sorting (FACS). FACS employs aplurality of color channels, low angle and obtuse light-scatteringdetection channels, and impedance channels, among other moresophisticated levels of detection, to separate or sort cells (see U.S.Pat. No. 5,061,620). Any of the single domain antibodies that bindmesothelin, as disclosed herein, can be used in these assays. Thus, theantibodies can be used in a conventional immunoassay, including, withoutlimitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Westernblot or imunoprecipitation.

EXAMPLES Example 1: Methods to Assess Binding and Cytotoxic Activitiesof Several Exemplary MSLN Targeting Trispecific Antigen Binding Proteins

Protein Production

Sequences of trispecific molecules were cloned into mammalian expressionvector pCDNA 3.4 (Invitrogen) preceded by a leader sequence and followedby a 6× Histidine Tag (SEQ ID NO: 379). Expi293F cells (LifeTechnologies A14527) were maintained in suspension in Optimum GrowthFlasks (Thomson) between 0.2 to 8×1e6 cells/ml in Expi 293 media.Purified plasmid DNA was transfected into Expi293 cells in accordancewith Expi293 Expression System Kit (Life Technologies, A14635)protocols, and maintained for 4-6 days post transfection. The amount ofthe exemplary trispecific proteins being tested, in the conditionedmedia, from the transfected Expi293 cells was quantitated using an Octetinstrument with Protein A tips and using a control trispecific proteinfor a standard curve.

Cytotoxicity Assays

A human T-cell dependent cellular cytotoxicity (TDCC) assay was used tomeasure the ability of T cell engagers, including trispecific molecules,to direct T cells to kill tumor cells (Nazarian et al. 2015. J BiomolScreen. 20:519-27). In this assay, T cells and target cancer cell linecells are mixed together at a 10:1 ratio in a 384 wells plate, andvarying amounts of the trispecific proteins being tested are added. Thetumor cell lines are engineered to express luciferase protein. After 48hours, to quantitate the remaining viable tumor cells, Steady-Glo®Luminescent Assay (Promega) was used.

In the instant study, titrations of conditioned media was added to TDCCassays (T cell Dependent Cell Cytotoxicity assays) to assess whether theanti-MSLN single domain antibody was capable of forming a synapsebetween T cells and a mesothelin expressing ovarian cancer cell line,OVCAR8. Viability of the OVCAR8 cells was measured after 48 hours. Itwas seen that the trispecific proteins mediated T cell killing. FIG. 2shows an example cell viability assay with test trispecific proteins 2A2and 2A4. The EC₅₀ for the TDCC activity of the test trispecific proteinsare listed below in Table 1.

TABLE 1 TDCC Activity of MSLN targeting trispecific proteins (TriTAC)Anti-MSLN Average TriTAC EC₅₀ [M] 2A2 1.6E−12 2A4 1.9E−09 11F3 2.2E−125D4 1.0E−09 9H2 1.1E−12 5C2 1.5E−12 5G2 3.6E−09 10B3 1.4E−12 2F4 7.3E−132C2 9.5E−09 5F2 5.3E−12 7C4 1.0E−08 7F1 2.4E−12 5D2 1.4E−11 6H2 2.0E−092D1 5.2E−11 12C2 8.0E−13 3F2 2.4E−08 1H2 2.5E−08 6F3 8.2E−10 2A1 1.2E−093G1 4.0E−09 12D1 1.1E−09 5H1 5.9E−12 4A2 1.7E−09 3B4 1.8E−12 7H2 5.5E−129F3 >1E−7  9B1 >1E−7 

Furthermore, it was observed that the TDCC activity of the MSLNtargeting trispecific proteins being tested was specific to mesothelinexpressing cells, because the trispecific proteins being tested did notmediate T cell killing of LNCaP cells, which do not express mesothelin.The trispecific proteins 2A2, 11F3, 9H2, 5C2, 10B3, 2F4, 5F2, 7F1, 2F4,5H1, 3B4, and 7H2, in particular did not show ant TDCC activity with theLnCaP cells.

Example 2: Xenograft Tumor Model

The MSLN targeting trispecific proteins of the previous example isevaluated in a xenograft model.

Female immune-deficient NOD/scid mice are sub-lethally irradiated (2 Gy)and subcutaneously inoculated with 1×10⁶ NCI-H28 cells into their rightdorsal flank. When tumors reach 100 to 200 mm³, animals are allocatedinto 3 treatment groups. Groups 2 and 3 (8 animals each) areintraperitoneally injected with 1.5×10⁷ activated human T-cells. Threedays later, animals from Group 3 are subsequently treated with a totalof 9 intravenous doses of 50 μg MSLN trispecific antigen-binding proteinof Example 1 (qdx9d). Groups 1 and 2 are only treated with vehicle. Bodyweight and tumor volume are determined for 30 days.

It is expected that animals treated with the MSLN targeting trispecificproteins of the previous examples have a statistically significant delayin tumor growth in comparison to the respective vehicle-treated controlgroup.

Example 3: Proof-of-Concept Clinical Trial Protocol for Administrationof the MSLN Trispecific Antigen-Binding Protein of Example 1 to OvarianCancer Patients

This is a Phase I/II clinical trial for studying the MSLN trispecificantigen-binding protein of Example 1 as a treatment for Ovarian Cancer.

Study Outcomes:

Primary:

Maximum tolerated dose of MSLN targeting trispecific proteins of theprevious examples

Secondary:

To determine whether in vitro response of MSLN targeting trispecificproteins of is the previous examples are associated with clinicalresponse

Phase I

The maximum tolerated dose (MTD) will be determined in the phase Isection of the trial.

-   -   1.1 The maximum tolerated dose (MTD) will be determined in the        phase I section of the trial.    -   1.2 Patients who fulfill eligibility criteria will be entered        into the trial to MSLN targeting trispecific proteins of the        previous examples.    -   1.3 The goal is to identify the highest dose of MSLN targeting        trispecific proteins of the previous examples that can be        administered safely without severe or unmanageable side effects        in participants. The dose given will depend on the number of        participants who have been enrolled in the study prior and how        well the dose was tolerated. Not all participants will receive        the same dose.

Phase II

-   -   2.1 A subsequent phase II section will be treated at the MTD        with a goal of determining if therapy with therapy of MSLN        targeting trispecific proteins of the previous examples results        in at least a 20% response rate.    -   Primary Outcome for the Phase II—To determine if therapy of MSLN        targeting trispecific proteins of the previous examples results        in at least 20% of patients achieving a clinical response (blast        response, minor response, partial response, or complete        response)

Eligibility:

-   -   Histologically confirmed ovarian cancer according to the current        World Health Organisation Classification, 2014        -   Surface epithelial—stromal tumors        -   Sex cord—stromal tumors        -   Germ cell tumors        -   Malignant, not otherwise specified        -   Age ≥18 yrs        -   Life expectancy ≥6 weeks

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Example 4: MH6T TriTAC Directs T Cells to Kill MSLN Expressing OvarianCancer Cells

A human T-cell dependent cellular cytotoxicity (TDCC) assay was used tomeasure the ability of T cell engagers, including trispecific molecules,to direct T cells to kill tumor cells (Nazarian et al. 2015. J BiomolScreen. 20:519-27). The Caov3 cells used in this assay were engineeredto express luciferase. T cells from 5 different healthy donors (donor02, donor 86, donor 41, donor 81, and donor 34) and target cancer cellsCaov3 were mixed together and varying amounts of an exemplarytrispecific molecule of this disclosure, MH6T TriTAC (SEQ ID NO: 98) wasadded and the mixture was incubated for 48 hours at 37° C. Caov3 cellsand T cells were also incubated for 48 hours at 37° C. with a controltrispecific molecule, GFP TriTAC (SEQ ID NO: 99), which targets GFP.After 48 hours, the remaining viable tumor cells were quantified by aluminescence assay.

It was observed that the MH6 TriTAC molecule was able to direct the Tcells from all 5 healthy donors to kill the target cancer cells Caov3(as shown in FIG. 3), whereas the control GFP TriTAC molecule was notable to direct the T cells from any of the 5 health donors to kill theCaov3 cells (also shown in FIG. 3).

A further assay, using the same protocol as described above, was carriedout using OVCAR3 cells. It was observed that the MH6 TriTAC molecule wasable to direct the T cells from all 5 healthy donors to kill the targetcancer cells OVCAR3 (as shown in FIG. 4), whereas the control GFP TriTACmolecule was not able to direct the T cells from any of the 5 healthdonors to kill the OVCAR3 cells (also shown in FIG. 4).

The EC₅₀ values for killing of MSLN expressing target cells are listedbelow in Table II.

TABLE II EC₅₀ values for MH6T TriTAC directed killing of MSLN-expressing ovarian cancer cell lines by T cells from 5 different healthydonors. Represented graphs of the raw data are provided in FIGS. 3 and4. EC₅₀ values (M) Donor02 Donor86 Donor41 Donor81 Donor35 Caov3 6.0E−136.8E−13 3.9E−13 5.9E−13 4.6E−13 Caov4 7.3E−12 1.1E−11 3.7E−12 4.7E−122.2E−12 OVCAR3 1.6E−12 2.5E−12 1.4E−12 1.6E−12 1.3E−12 OVCAR8 2.2E−123.2E−12 1.4E−12 1.9E−12 1.7E−12

Example 5: MH6T TriTAC Directs T Cells to Kill Cells Expressing MSLN butnot Cells that do not Express MSLN

In this assay, T cells from a healthy donor was incubated with targetcancer cells that express MSLN (Caov3 cells, Caov4 cells, OVCAR3 cells,and OVCAR8 cells) or target cancer cells that do not express MSLN(NCI-H510A cells, MDAPCa2b cells). Each of the target cells used in thisstudy were engineered to express luciferase. Varying amounts of the MH6TTriTAC (SEQ ID NO: 98) molecule was added to the mixture of T cells andtarget cancer cells listed above. The mixture was incubated for 48 hoursat 37° C. After 48 hours, the remaining viable target cancer cells werequantified using a luminescent assay.

It was observed that the MH6 TriTAC molecule was able to direct T cellsto kill MSLN expressing target cancer cells (i.e., Caoc3, Caov4, OVCAR3,and OVCAR8 cells, as shown in FIG. 5). However, the MH6T TriTAC moleculewas not able to direct T cells to kill MSLN non-expressing target cancercells (MDAPCa2b and NCI-H510A cells), also shown in FIG. 5.

The EC₅₀ values for killing of MSLN expressing cancer cells are listedbelow in Table III.

TABLE III EC₅₀ values for MH6T TriTAC directed T cell killing ofMSLN-expressing cancer cell lines. EC₅₀ MSLN sites Tumor origin CellLine (pM) per cell Ovarian Caov3 0.6 51262 Caov4 7.3 101266 OVCAR3 1.640589 OVCAR8 2.2 40216 SKOV3 3.6 10617 Pancreatic Hs766T 7.8 5892 CaPan23.2 27413 HPaFII 15 17844 NSCLC NCI-H596 1.5 103769 NCI-H292 3.8 5977NCI-H1563 2.6 17221 Mesothelioma NCI-H2052 8.0 not determined NCI-H24522.3 not determined Engineered HEK293 expressing human 0.9 128091(non-tumor) MSLN HEK293 293 expressing 0.7 140683 cynomolgus MSLN

Example 6: MH6T TriTAC Directed T Cells from Cynomolgus Monkeys to KillHuman Ovarian Cancer Cell Lines

In this assay, peripheral blood mononuclear cells (PBMCs; T cells are acomponent of the PBMCs) from a cynomolgus monkey donor was mixed withtarget cancer cells that express MSLN (CaOV3 cells and OVCAR3 cells) andvarying amounts of the MH6T TriTAC molecule (SEQ ID NO: 98) was added tothe mixture, and incubated for 48 hours at 37° C. In parallel, a mixtureof cynomolgus PBMCs and MSLN expressing cells, as above, were incubatedwith varying amounts of a control TriTAC molecule GFP TriTAC (SEQ ID NO:99) that targets GFP, for 48 hours at 37° C. Target cancer cells used inthis assay were engineered to express luciferase. After 48 hours, theremaining viable target cells were quantified using a luminescenceassay.

It was observed that the MH6 TriTAC molecule was able to efficientlydirect cynomolgus PBMCs to kill MSLN expressing cells (i.e., Caov3 andOVCAR, as shown in FIG. 6, whereas the control GFP TriTAC molecule wasnot able to direct the cynomolgus PBMCs to kill the cells (also shown inFIG. 6). The EC₅₀ values for the MH6T TriTAC molecule was 2.9 pM forOVCAR3 cells and 3.0 pM for Caov3 cells, which were not significantlydifferent that EC₅₀ values observed with human T cells, as shown inTable II.

Example 7: MH6T TriTAC Molecule Directed Killing of MSLN-ExpressingNCI-H2052 Mesothelioma Cells by T Cells in the Presence or Absence ofHuman Serum Albumin

The aim of this study was to assess if binding to human serum albumin(HSA) by MH6T TriTAC molecule impacted the ability of the MH6T TriTACmolecule to direct T cells to kill MSLN-expressing cells. NCI-H2052mesothelioma cells used in this study were engineered to expressluciferase. T cells from a healthy donor and MSLN expressing cells(NCI-H2052) were mixed and varying amounts of the MH6T TriTAC (SEQ IDNO: 98) molecule was added to the mixture. The mixture was incubated for48 hours at 37° C., in presence or absence of HSA. A mixture ofNCI-H2052 cells and T cells were also incubated for 48 hours at 37° C.with a control trispecific molecule, GFP TriTAC (SEQ ID NO: 99), whichtargets GFP, in presence or absence of HSA. After 48 hours, theremaining viable target cells were quantified using a luminescenceassay.

It was observed that the MH6 TriTAC molecule was able to efficientlydirect T cells to kill NCI-H2052 cells (as shown in FIG. 7) in presenceor absence of HSA, whereas the control GFP TriTAC molecule was not ableto do that (also shown in FIG. 7). It was also observed that in presenceof HSA, the EC₅₀ value for cell killing was increased by about 3.2 folds(as shown in Table IV).

Further TDCC assays were carried out with the MH6T TriTAC molecule, inpresence or absence of 15 mg/ml HSA, with additional MSLN-expressingcells lines and the EC₅₀ values are presented in Table IV.

TABLE IV EC₅₀ values for MH6T TriTAC directed killing of MSLN-expressingcancer cells by T cells in the presence or absence of HSA EC₅₀ no EC₅₀with EC₅₀ shift Cell line HSA (pM) HSA (pM) (fold) OVCAR8 2.7 8.7 3.2SKOV3 3.9 11 2.8 NCI-H2052 8.0 26 3.2 NCI-H24522 2.3 6.3 2.7 Caov3 0.83.6 4.3 OVCAR3 1.6 3.8 2.4

Example 8: T Cells from 4 Different Donors Secrete TNF-Alpha in thePresence of MH6T TriTAC and MSLN-Expressing Caov4 Cells

The target cancer cells CaOv4 used in this assay were engineered toexpress luciferase. In this assay, T cells from 4 different healthydonors (donor 02, donor 86, donor 35, and donor 81) and Caov4 cells weremixed together and varying amounts of the MH6T TriTAC molecule (SEQ IDNO: 98) was added and the mixture was incubated for 48 hours at 37° C.Caov4 cells and T cells were also incubated for 48 hours at 37° C. witha control trispecific molecule, GFP TriTAC (SEQ ID NO: 99), whichtargets GFP. Conditioned medium from the TDCC assay was collected at 48hours, before measuring the target cancer cell viability, using aluminescence assay. The concentration of TNF-α in the conditioned mediumwas measured using an AlphaLISA assay kit (Perkin Elmer).

It was observed that TNF-α was secreted into the medium in presence ofCaov4 cells and the MH6T TriTAC molecule but not in presence of Caov4cells and the control GFP TriTAC molecule, as shown in FIG. 8.

Furthermore, efficient killing was observed with T cells from all 4healthy donors, in presence of the MH6T TriTAC molecule, but not inpresence of the control GFP TriTAC molecule. TDCC assays were also setup for additional MSLN expressing cell lines (Caov3 cells, OVCAR3 cells,and OVCAR8 cells) and similar TNF-α expression was observed. The EC₅₀values for MH6T TriTAC induced expression of TNF-α are presented inTable V. However, when the assay was carried out using cancer cells thatdo not express MSLN (NCI-H510A cells, or MDAPCa2b cells), no MH6T TriTACdirected secretion of TNF-α was observed (data not shown). Thus, thisstudy demonstrated that the MH6T TriTAC molecule was able to activate Tcells in the presence of MSLN-expressing target cancer cells.

TABLE V EC₅₀ values for MH6T TriTAC molecule induced expression of TNF-αby T cells from 4 different T cell donors and 4 differentMSLN-expressing cell lines TNFα EC₅₀ values (M) MH6T MH6T MH6T MH6TTriTAC TriTAC TriTAC TriTAC Donor 2 Donor 86 Donor 35 Donor 81 Caov35.2E−12 5.4E−12 5.9E−12 4.9E−12 Caov4 7.2E−12 6.0E−12 5.5E−12 5.5E−12OVCAR3 9.2E−12 4.0E−12 1.7E−11 8.9E−12 OVCAR8 1.3E−11 9.1E−12 5.1E−125.0E−12

Example 9: Activation of CD69 Expression on T Cells from 4 DifferentDonors in Presence of MH6T TriTAC and MSLN-Expressing OVCAR8 Cells

The OVCAR8 cells used in this assay were engineered to expressluciferase. In this assay, T cells from 4 different healthy donors(donor 02, donor 86, donor 35, and donor 81) and OVCAR8 cells were mixedtogether and varying amounts of the MH6T TriTAC molecule (SEQ ID NO: 98)was added and the mixture was incubated for 48 hours at 37° C. OVCAR8cells and T cells were also incubated for 48 hours at 37° C. with acontrol trispecific molecule, GFP TriTAC (SEQ ID NO: 99), which targetsGFP. After 48 hours, T cells were collected, and CD69 expression on theT cells was measured by flow cytometry.

CD69 expression was detected on T cells from all 4 healthy donors inpresence of OVCAR8 cells and the MH6T TriTAC molecule but not inpresence of the negative control GFP TriTAC and OVCAR8 cells, as shownin FIG. 9. TDCC assays were also set up for additional MSLN expressingcells (Caov3 cells, OVCAR3 cells, and OVCAR8 cells) and similar CD69expression was observed. The EC₅₀ values for MH6T TriTAC inducedactivation of CD69 in Caov3 cells and OVCAR8 cells are presented inTable VI.

TABLE VI EC50 values for activation of CD69 expression on T cells from 4different donors in presence of MH6T TriTAC molecule and MSLN-expressingOVCAR8 cells or Caov3 cells. EC₅₀ table Caov3 CD69 (M) OVCAR8 CD69 (M)Donor 35 ~1.5E−13  1.4E−13 Donor 2 2.5E−13 4.2E−13 Donor 81 2.5E−132.5E−13 Donor 86 3.7E−13 3.7E−13

When the assay was carried out using cancer cells that do not expressMSLN (NCI-H510A cells or MDAPCa2b cells), no MH6T induced activation ofCD69 was observed (data not shown). Thus, this study demonstrated thatthe MH6T TriTAC molecule was able to activate T cells in the presence ofMSLN-expressing target cancer cells.

Example 10: Measurement of MH6T TriTAC Binding to MSLNExpressing/Non-Expressing Cell Lines

For this study, certain target cancer cells that express MSLN (Caov3cells, CaOV4 cells, OVCAR3 cells, and OVCAR8 cells) and certain cancercells that do not express MSLN (MDAPCa2b cells, and NCI-H510A cells)were incubated with the MH6 TriTAC molecule (SEQ ID NO: 98) or a controlGFP TriTAC molecule (SEQ ID NO: 99). Following incubation, the cellswere washed to remove unbound MH6T or GFP TriTAC molecules and furtherincubated with a secondary antibody conjugated to Alexa Fluor 647, whichis able to recognize the anti-albumin domain in the TriTAC molecules.Binding of the MH6T TriTAC or that of GFP TriTAC to the MSLN expressingor MSLN non-expressing cells was measured by flow cytometry.

Robust binding of the MH6T TriTAC molecule to cell lines that expressMSLN (Caov3, Caov4, OVCAR3, and OVCAR8) was observed, as seen in FIG.10A (top left panel shows binding of MH6T TriTAC to Caov3 cells; topright panel shows binding of MH6T TriTAC to Caov4 cells; bottom leftpanel shows binding of MH6T TriTAC to OVCAR3 cells; bottom right panelshows binding of MH6T TriTAC to OVCAR8 cells); and no binding wasobserved in cell lines that do not express MSLN (left panel shows lackof binding of MH6T TriTAC to MDAPCa2b cells and the right panel showslack of binding of MH6T TriTAC to NCI-H510A cells), as shown in FIG.10B. Furthermore, no binding was observed when any of the cell typeswere incubated with the GFP TriTAC molecule, as shown in both FIGS. 10Aand 10B.

Example 11: Measurement of MH6T TriTAC Binding to T Cells from Donors

For this study, T cells from 4 health donors were incubated with the MH6TriTAC molecule (SEQ ID NO: 98) or a buffer, as negative control.Following incubation, the cells were washed to remove unbound MH6TTriTAC molecules and further incubated with an Alexa Fluor 647conjugated secondary antibody, which is able to recognize theanti-albumin domain in the MH6T TriTAC molecule. Binding of the MH6TTriTAC to the cells was measured by flow cytometry.

Robust binding of the MH6T TriTAC was observed to T cells from all fourdonors, treated with the MH6T TriTAC molecule, as shown in FIG. 11 (topleft panel shows binding of MH6T TriTAC to T cells from donor 2; topright panel shows binding of MH6T TriTAC to T cells from donor 35;bottom left panel shows binding of MH6T TriTAC to T cells from donor 41;bottom right panel shows binding of MH6T TriTAC to T cells from donor81).

Example 12: Inhibition of Tumor Growth in Mice Treated with MH6T TriTACMolecule

For this study, 10⁷ NCI-H292 cells and 10⁷ human PBMCs were co-implantedsubcutaneously in two groups of NCG mice (8 mice per group). After 5days, mice in one group were injected with the MH6T TriTAC molecule (SEQID NO: 98), daily for 10 days (days 5-14) at a dose of 0.25 mg/kg; andmice in the other group were injected with a vehicle control. Tumorvolumes were measured after every few days and the study was terminatedat day 36. Significant inhibition of tumor growth was observed in themice injected with the MH6 TriTAC molecules, compared to those injectedwith the vehicle control, as shown in FIG. 12.

Example 13: Pharmacokinetics of MH6T TriTAC in Cynomolgus Monkeys

For this study, two cynomolgus monkeys were injected with 10 mg/kg doseof MH6T TriTAC molecule (SEQ ID NO: 98), intravenously, and serumsamples were collected at various time points after the injection. Theamount of the MH6T TriTAC in the serum was measured using anti-idiotypeantibodies recognizing the MH6T TriTAC molecule, in anelectrochemiluminescient assay. FIG. 13 shows a plot for the serum M6HTTriTAC levels at various time points. The data was then used tocalculate the pharmacokinetic properties of MH6T TriTAC molecule, asprovided in

TABLE VII Pharmacokinetic parameters for MH6T TriTAC Terminal C_(max)AUC, 0-inf Clearance Vss Dose Level t_(1/2) (nM) (hr*nM) (mL/hr/kg)(mL/kg) 10 mg/kg 112 6,130 355,000 0.58 70.0

Example 14: Optimization of CD3ε Binding Affinity for Maximum Activityand Exposure of Two Exemplary Trispecific Molecules of this Disclosure

CD3ε, MSLN, and albumin binding affinities of two exemplary trispecificmolecules of this disclosure, TriTAC 74 (SEQ ID NO: 100) and TriTAC 75(SEQ ID NO: 101) were measured for this study. It was observed thatTriTAC74 was about 5 times more potent in binding human CD3 thanTriTAC75, even though the binding affinities of the two molecules weresimilar for the tumor target (MSLN) and albumin, as shown in FIG. 14.Additionally, TDCC assays were carried out with the TriTAC 74 and TriTAC75 molecules, using SKOV3 and OVCAR cells. FIG. 14 shows the EC₅₀ valuesobtained in the TDCC assays.

The difference in CD3ε affinity was found to lead to approximately 30%to 50% increase in AUC, in TriTAC 74, compared to TriTAC 75, as measuredin a pharmacokinetic assay after injecting cynomolgus monkeys with theTriTAC molecules (at an intravenous bolus dose of 0.02 mg/kg), providedin Table VIII. Serum levels of the TriTAC molecules were measured atvarious time points after the injection, using a Meso Scale Discovery(MSD) assay with anti-idiotype antibodies. The MSD assay was carried outusing n=2 replicates. Serum concentrations observed in MSD assay areshown in FIG. 15 and the pharmacokinetic parameters are listed in TableVIII.

TABLE VIII Pharmacokinetic parameters for TriTAC 74 and TriTAC 75Terminal AUC, 0-last AUC, 0-inf Clearance Vss TriTAC t_(1/2) (hr*nM)(hr*nM) (mL/hr/kg) (mL/kg) 74 84.9 1030 1050 0.367 36.8 75 89.4 715 7270.522 54.2

Sequence Table Exemplary MSLN binding Sequence trispecific ID No.protein Sequence SEQ ID 9B1QVQLVESGGGLVQPGGSLRLSCAASGRTFSVRGMAWYRQAGNNRALVATMNPDGFPN NO: 1YADAVKGRFTISWDIAENTVYLQMNSLNSEDTTVYYCNSGPYWGQGTQVTVSS SEQ ID 9F3QVQLVESGGGLVQAGGSLRLSCAASGSIPSIEQMGWYRQAPGKQRELVAALTSGGRA NO: 2NYADSVKGRFTISGDNVRNMVYLQMNSLKPEDTAIYYCSAGRFKGDYAQRSGMDYWG KGTLVTVSSSEQ ID 7H2 QVQLVESGGGLVQAGGSLRLSCAFSGTTYTFDLMSWYRQAPGKQRTVVASISSDGRTNO: 3 SYADSVRGRFTISGENGKNTVYLQMNSLKLEDTAVYYCLGQRSGVRAFWGQGTQVTV SSSEQ ID 3B4 QVQLVESGGGLVQAGGSLRLSCVASGSTSNINNMRWYRQAPGKERELVAVITRGGYANO: 4 IYLDAVKGRFTISRDNANNAIYLEMNSLKPEDTAVYVCNADRVEGTSGGPQLRDYFGQGTQVTVSS SEQ ID 4A2QVQLVESGGGLVQAGGSLRLSCAASGSTFGINAMGWYRQAPGKQRELVAVISRGGST NO: 5NYADSVKGRFTISRDNAENTVSLQMNTLKPEDTAVYFCNARTYTRHDYWGQGTQVTV SS SEQ ID 12D1QVRLVESGGGLVQAGGSLRLSCAASISAFRLMSVRWYRQDPSKQREWVATIDQLGRT NO: 6NYADSVKGRFAISKDSTRNTVYLQMNMLRPEDTAVYYCNAGGGPLGSRWLRGRHWGQ GTQVTVSSSEQ ID 3G1 QVRLVESGGGLVQAGESLRLSCAASGRPFSINTMGWYRQAPGKQRELVASISSSGDFNO: 7 TYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNARRTYLPRRFGSWGQGTQ VTVSSSEQ ID 2A1 QVQPVESGGGLVQPGGSLRLSCVVSGSDFTEDAMAWYRQASGKERESVAFVSKDGKRNO: 8 ILYLDSVRGRFTISRDIDKKTVYLQMDNLKPEDTGVYYCNSAPGAARNYWGQGTQVT VSSSEQ ID 6F3 QVQPVESGGGLVQPGGSLRLSCVVSGSDFTEDAMAWYRQASGKERESVAFVSKDGKRNO: 9 ILYLDSVRGRFTISRDIYKKTVYLQMDNLKPEDTGVYYCNSAPGAARNVWGQGTQVT VSSSEQ ID 1H2 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDNO: 10 TLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVS SSEQ ID 3F2 QVQIVESGGGLVQAGGSLRLSCVASGLTYSIVAVGWYRQAPGKEREMVADISPVGNTNO: 11 NYADSVKGRFTISKENAKNTVYLQMNSLKPEDTAVYYCHIVRGWLDERPGPGPIVYWGQGTQVTVSS SEQ ID 12C2QVQLVESGGGLVQTGGSLRLSCAASGLTFGVYGMEWFRQAPGKQREWVASHTSTGYV NO: 12YYRDSVKGRFTISRDNAKSTVYLQMNSLKPEDTAIYYCKANRGSYEYWGQGTQVTVS S SEQ ID 2D1QVQLVESGGGLVQAGGSLRLSCAASTTSSINSMSWYRQAQGKQREPVAVITDRGSTS NO: 13YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYTCHVIADWRGYWGQGTQVTVSS SEQ ID 6H2QVQLVESGGGLVQAGGSLRLSCAASGRTLSRYAMGWFRQAPGKERQFVAAISRSGGT NO: 14TRYSDSVKGRFTISRDNAANTFYLQMNNLRPDDTAVYYCNVRRRGWGRTLEYWGQGT QVTVSS SEQ ID5D2 QVQLGESGGGLVQAGGSLRLSCAASGSIFSPNAMIWHRQAPGKQREPVASINSSGST NO: 15NYGDSVKGRFTVSRDIVKNTMYLQMNSLKPEDTAVYYCSYSDFRRGTQYWGQGTQVT VSS SEQ ID 7C4QVQLVESGGGLVPSGGSLRLSCAASGATSAITNLGWYRRAPGQVREMVARISVREDK NO: 16EDYEDSVKGRFTISRDNTQNLVYLQMNNLQPHDTAIYYCGAQRWGRGPGTTWGQGTQ VTVSS SEQ ID5F2 QVQLVESGGGLVQAGGSLRLSCAASGSTFRIRVMRWYRQAPGTERDLVAVISGSSTY NO: 17YADSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCNADDSGIARDYWGQGTQVTV SS SEQ ID 2C2QVQLVESGGGLVQAGESRRLSCAVSGDTSKFKAVGWYRQAPGAQRELLAWINNSGVG NO: 18NTAESVKGRFTISRDNAKNTVYLQMNRLTPEDTDVYYCRFYRRFGINKNYWGQGTQV TVSS SEQ ID5G2 QVQLVESGGGLVQAGGSLRLSCAASGSTFGNKPMGWYRQAPGKQRELVAVISSDGGS NO: 19TRYAALVKGRFTISRDNAKNTVYLQMESLVAEDTAVYYCNALRTYYLNDPVVFSWGQ GTQVTVSSSEQ ID 9H2 QVQLVESGGGLVQAGGSLRLSCAASGSTSSINTMYWYRQAPGKERELVAFISSGGSTNO: 20 NVRDSVKGRFSVSRDSAKNIVYLQMNSLTPEDTAVYYCNTYIPLRGTLHDYWGQGTQ VTVSSSEQ ID 5D4 QVQLVESGGGLVQAGGSLRLSCVASGRTDRITTMGWYRQAPGKQRELVATISNRGTSNO: 21 NYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNARKWGRNYWGQGTQVTVS SSEQ ID 2A4 QVQLVESGGGLVQARGSLRLSCTASGRTIGINDMAWYRQAPGNQRELVATITKGGTTNO: 22 DYADSVDGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTKRREWAKDFEYWGQGTQ VTVSSSEQ ID 7F1 QVQLVESGGGLVQAGGSLRLSCAASAIGSINSMSWYRQAPGKQREPVAVITDRGSTSNO: 23 YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYTCHVIADWRGYWGQGTQVTVSS SEQ ID5C2 QVQLVESGGGLVQAGGSLRLSCAASGSTSSINTMYWFRQAPGEERELVATINRGGST NO: 24NVRDSVKGRFSVSRDSAKNIVYLQMNRLKPEDTAVYYCNTYIPYGGTLHDFWGQGTQ VTVSS SEQ ID2F4 QVQLVESGGGLVQAGGSLRLSCTTSTTFSINSMSWYRQAPGNQREPVAVITNRGTTS NO: 25YADSVKGRFTISRDNARNTVYLQMDSLKPEDTAIYTCHVIADWRGYWGQGTQVTVSS SEQ ID 2A2QVQLVESGGGLVQAGGSLTLSCAASGSTFSIRAMRWYRQAPGTERDLVAVIYGSSTY NO: 26YADAVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCNADTIGTARDYWGQGTQVTV SS SEQ ID 11F3QVQLVESGGGLVQAGGSLRLSCVASGRTSTIDTMYWHRQAPGNERELVAYVTSRGTS NO: 27NVADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCSVRTTSYPVDFWGQGTQVT VSS SEQ ID10B3 QVQLVESGGGLVQAGGSLRLSCAASGSTSSINTMYWYRQAPGKERELVAFISSGGST NO: 28NVRDSVKGRFSVSRDSAKNIVYLQMNSLKPEDTAVYYCNTYIPYGGTLHDFWGQGTQ VTVSS SEQ ID5H1 QVQLVESGGGLVQPGGSLRLSCAASGGDWSANFMYWYRQAPGKQRELVARISGRGVV NO: 29DYVESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVASYWGQGTQVTVSS SEQ ID MH1EVQLVESGGGLVQPGGSLRLSCAASGGDWSANFMYWYRQAPGKQRELVARISGRGVV NO: 30(exemplary DYVESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVASYWGQGTLVTVSShumanized version of 5H1) SEQ ID MH2EVQLVESGGGLVQPGGSLRLSCAASGGDWSANFMYWVRQAPGKGLEWVSRISGRGVV NO: 31(exemplary DYVESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVASYWGQGTLVTVSShumanized version of 5H1) SEQ ID MH3EVQLVESGGGLVQAGGSLRLSCAASGSTSSINTMYWYRQAPGKERELVAFISSGGST NO: 32(exemplary NVRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNTYIPYGGTLHDFWGQGTLhumanized VTVSS version of 10B3) SEQ ID MH4EVQLVESGGGLVQPGGSLRLSCAASGSTSSINTMYWYRQAPGKERELVAFISSGGST NO: 33(exemplary NVRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNTYIPYGGTLHDFWGQGTLhumanized VTVSS version of 10B3) SEQ ID MH5EVQLVESGGGLVQPGGSLRLSCAASGSTSSINTMYWVRQAPGKGLEWVSFISSGGST NO: 34(exemplary NVRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNTYIPYGGTLHDFWGQGTLhumanized VTVSS version of 10B3) SEQ ID MH6-GGQVQLVESGGGVVQAGGSLRLSCAASGSTFSIRAMRWYRQAPGTERDLVAVIYGSSTY NO: 35(exemplary YADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNADTIGTARDYWGQGTLVTVhumanized SSGG version of 2A2) SEQ ID MH7-GGQVQLVESGGGVVQPGGSLRLSCAASGSTFSIRAMRWYRQAPGKERELVAVIYGSSTY NO: 36(exemplary YADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNADTIGTARDYWGQGTLVTVhumanized SSGG version of 2A2) SEQ ID MH8-GGQVQLVESGGGVVQPGGSLRLSCAASGSTFSIRAMRWVRQAPGKGLEWVSVIYGSSTY NO: 37(exemplary YADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNADTIGTARDYWGQGTLVTVhumanized SSGG version of 2A2) SEQ ID MH9EVQLVESGGGLVQAGGSLRLSCVASGRTSTIDTMYWHRQAPGNERELVAYVTSRGTS NO: 38(exemplary NVADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSVRTTSYPVDFWGQGTLVThumanized VS version of 11F3) SEQ ID MH10EVQLVESGGGLVQPGGSLRLSCAASGRTSTIDTMYWHRQAPGKERELVAYVTSRGTS NO: 39(exemplary NVADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSVRTTSYPVDFWGQGTLVThumanized VSS version of 11F3) SEQ ID MH11EVQLVESGGGLVQPGGSLRLSCAASGRTSTIDTMYWVRQAPGKGLEWVSYVTSRGTS NO: 40(exemplary NVADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSVRTTSYPVDFWGQGTLVThumanized VSS version of 11F3) SEQ ID Exemplary ESGGGLV NO: 41 conservedregion in MSLN binding domain SEQ ID Exemplary LSC NO: 42 conservedregion in MSLN binding domain SEQ ID Exemplary GRF NO: 43 conservedregion in MSLN binding domain SEQ ID Exemplary VTVSS NO: 44 conservedregion in MSLN binding domain SEQ ID Exemplary QLVESGGG NO: 45 conservedregion in MSLN binding domain SEQ ID Exemplary GGSLRLSCAASG NO: 46conserved region in MSLN binding domain SEQ ID Exemplary ASG NO: 47conserved region in MSLN binding domain SEQ ID Exemplary RQAPG NO: 48conserved region in MSLN binding domain SEQ ID ExemplaryVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC NO: 49 conserved region in MSLNbinding domain SEQ ID Exemplary WGQGTLVTVSS NO: 50 conserved region inMSLN binding domain SEQ ID Exemplary GRTFSVRGMA NO: 51 CDR1 of MSLNbinding domain SEQ ID Exemplary INSSGSTNYG NO: 52 CDR2 of MSLN bindingdomain SEQ ID Exemplary NAGGGPLGSR NO: 53 CDR3 of MSLN binding domainSEQ ID Exemplary GGDWSANFMY NO: 54 CDR1 of MSLN binding domain SEQ IDExemplary ISSGGSTNVR NO: 55 CDR2 of MSLN binding domain SEQ ID ExemplaryNADTIGTARD NO: 56 CDR3 of MSLN binding domain SEQ ID MesothelinMALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPN NO: 57 proteinISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPED sequenceLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLAS TLA SEQ ID9B1 TriTAC QVQLVESGGGLVQPGGSLRLSCAASGRTFSVRGMAWYRQAGNNRALVATMNPDGFPNNO: 58 YADAVKGRFTISWDIAENTVYLQMNSLNSEDTTVYYCNSGPYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 9F3 TriTACQVQLVESGGGLVQAGGSLRLSCAASGSIPSIEQMGWYRQAPGKQRELVAALTSGGRA NO: 59NYADSVKGRFTISGDNVRNMVYLQMNSLKPEDTAIYYCSAGRFKGDYAQRSGMDYWGKGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID7H2 TriTAC QVQLVESGGGLVQAGGSLRLSCAFSGTTYTFDLMSWYRQAPGKQRTVVASISSDGRTNO: 60 SYADSVRGRFTISGENGKNTVYLQMNSLKLEDTAVYYCLGQRSGVRAFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 3B4 TriTACQVQLVESGGGLVQAGGSLRLSCVASGSTSNINNMRWYRQAPGKERELVAVITRGGYA NO: 61IYLDAVKGRFTISRDNANNAIYLEMNSLKPEDTAVYVCNADRVEGTSGGPQLRDYFGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID4A2 TriTAC QVQLVESGGGLVQAGGSLRLSCAASGSTFGINAMGWYRQAPGKQRELVAVISRGGSTNO: 62 NYADSVKGRFTISRDNAENTVSLQMNTLKPEDTAVYFCNARTYTRHDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 12D1 TriTACQVRLVESGGGLVQAGGSLRLSCAASISAFRLMSVRWYRQDPSKQREWVATIDQLGRT NO: 63NYADSVKGRFAISKDSTRNTVYLQMNMLRPEDTAVYYCNAGGGPLGSRWLRGRHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 3G1 TriTACQVRLVESGGGLVQAGESLRLSCAASGRPFSINTMGWYRQAPGKQRELVASISSSGDF NO: 64TYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNARRTYLPRRFGSWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 2A1 TriTACQVQPVESGGGLVQPGGSLRLSCVVSGSDFTEDAMAWYRQASGKERESVAFVSKDGKR NO: 65ILYLDSVRGRFTISRDIDKKTVYLQMDNLKPEDTGVYYCNSAPGAARNYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 6F3 TriTACQVQPVESGGGLVQPGGSLRLSCVVSGSDFTEDAMAWYRQASGKERESVAFVSKDGKR NO: 66ILYLDSVRGRFTISRDIYKKTVYLQMDNLKPEDTGVYYCNSAPGAARNVWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 1H2 TriTACEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSD NO: 67TLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 3F2 TriTACQVQIVESGGGLVQAGGSLRLSCVASGLTYSIVAVGWYRQAPGKEREMVADISPVGNT NO: 68NYADSVKGRFTISKENAKNTVYLQMNSLKPEDTAVYYCHIVRGWLDERPGPGPIVYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID12C2 TriTAC QVQLVESGGGLVQTGGSLRLSCAASGLTFGVYGMEWFRQAPGKQREWVASHTSTGYVNO: 69 YYRDSVKGRFTISRDNAKSTVYLQMNSLKPEDTAIYYCKANRGSYEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 2D1 TriTACQVQLVESGGGLVQAGGSLRLSCAASTTSSINSMSWYRQAQGKQREPVAVITDRGSTS NO: 70YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYTCHVIADWRGYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 6H2 TriTACQVQLVESGGGLVQAGGSLRLSCAASGRTLSRYAMGWFRQAPGKERQFVAAISRSGGT NO: 71TRYSDSVKGRFTISRDNAANTFYLQMNNLRPDDTAVYYCNVRRRGWGRTLEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 5D2 TriTACQVQLGESGGGLVQAGGSLRLSCAASGSIFSPNAMIWHRQAPGKQREPVASINSSGST NO: 72NYGDSVKGRFTVSRDIVKNTMYLQMNSLKPEDTAVYYCSYSDFRRGTQYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 7C4 TriTACQVQLVESGGGLVPSGGSLRLSCAASGATSAITNLGWYRRAPGQVREMVARISVREDK NO: 73EDYEDSVKGRFTISRDNTQNLVYLQMNNLQPHDTAIYYCGAQRWGRGPGTTWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 5F2 TriTACQVQLVESGGGLVQAGGSLRLSCAASGSTFRIRVMRWYRQAPGTERDLVAVISGSSTY NO: 74YADSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCNADDSGIARDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 2C2 TriTACQVQLVESGGGLVQAGESRRLSCAVSGDTSKFKAVGWYRQAPGAQRELLAWINNSGVG NO: 75NTAESVKGRFTISRDNAKNTVYLQMNRLTPEDTDVYYCRFYRRFGINKNYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 5G2 TriTACQVQLVESGGGLVQAGGSLRLSCAASGSTFGNKPMGWYRQAPGKQRELVAVISSDGGS NO: 76TRYAALVKGRFTISRDNAKNTVYLQMESLVAEDTAVYYCNALRTYYLNDPVVFSWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 9H2 TriTACQVQLVESGGGLVQAGGSLRLSCAASGSTSSINTMYWYRQAPGKERELVAFISSGGST NO: 77NVRDSVKGRFSVSRDSAKNIVYLQMNSLTPEDTAVYYCNTYIPLRGTLHDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 5D4 TriTACQVQLVESGGGLVQAGGSLRLSCVASGRTDRITTMGWYRQAPGKQRELVATISNRGTS NO: 78NYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNARKWGRNYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 2A4 TriTACQVQLVESGGGLVQARGSLRLSCTASGRTIGINDMAWYRQAPGNQRELVATITKGGTT NO: 79DYADSVDGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTKRREWAKDFEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 7F1 TriTACQVQLVESGGGLVQAGGSLRLSCAASAIGSINSMSWYRQAPGKQREPVAVITDRGSTS NO: 80YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYTCHVIADWRGYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 5C2 TriTACQVQLVESGGGLVQAGGSLRLSCAASGSTSSINTMYWFRQAPGEERELVATINRGGST NO: 81NVRDSVKGRFSVSRDSAKNIVYLQMNRLKPEDTAVYYCNTYIPYGGTLHDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 2F4 TriTACQVQLVESGGGLVQAGGSLRLSCTTSTTFSINSMSWYRQAPGNQREPVAVITNRGTTS NO: 82YADSVKGRFTISRDNARNTVYLQMDSLKPEDTAIYTCHVIADWRGYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 2A2 TriTACQVQLVESGGGLVQAGGSLTLSCAASGSTFSIRAMRWYRQAPGTERDLVAVIYGSSTY NO: 83YADAVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCNADTIGTARDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 11F3 TriTACQVQLVESGGGLVQAGGSLRLSCVASGRTSTIDTMYWHRQAPGNERELVAYVTSRGTS NO: 84NVADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCSVRTTSYPVDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 10B3 TriTACQVQLVESGGGLVQAGGSLRLSCAASGSTSSINTMYWYRQAPGKERELVAFISSGGST NO: 85NVRDSVKGRFSVSRDSAKNIVYLQMNSLKPEDTAVYYCNTYIPYGGTLHDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID 5H1 TriTACQVQLVESGGGLVQPGGSLRLSCAASGGDWSANFMYWYRQAPGKQRELVARISGRGVV NO: 86DYVESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVASYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID Exemplary (GS)nNO: 87 linker sequence SEQ ID Exemplary (GGS)n NO: 88 linker sequenceSEQ ID Exemplary (GGGS)n NO: 89 linker sequence SEQ ID Exemplary (GGSG)nNO: 90 linker sequence SEQ ID Exemplary (GGSGG)n NO: 91 linker sequenceSEQ ID Exemplary (GGGGS)n NO: 92 linker sequence SEQ ID Exemplary(GGGGG)n NO: 93 linker sequence SEQ ID Exemplary (GGG)n NO: 94 linkersequence SEQ ID Exemplary (GGGGS)4 NO: 95 linker sequence SEQ IDExemplary (GGGGS)3 NO: 96 linker sequence SEQ ID Sortase LPETG NO: 97recognition domain SEQ ID MH6T TriTACQVQLVESGGGVVQAGGSLTLSCAASGSTFSIRAMRWYRQAPGTERDLVAVIYGSSTY NO: 98YADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNADTIGTARDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID GFP TriTACQVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDR NO: 99SSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID TriTAC 74QVQLVESGGGVVQAGGSLRLSCAASGSTFSIRAMRWYRQAPGTERDLVAVIYGSSTYYADAVK NO: 100GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNADTIGTARDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGNTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGDSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTHGNYPNWVQQKPGQAPRGLIGGTKVLAPGTPARFSGSLALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH SEQ IDTriTAC 75 QVQLVESGGGVVQAGGSLRLSCAASGSTFSIRAMRWYRQAPGTERDLVAVIYGSSTYNO: 101 YADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNADTIGTARDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLHHHHHH SEQ ID Anti-MSLN-QVQLVESGGGVVQAGGSLTLSCAASGSTFSIRAMRWYRQAPGTERDLVAVIYGSSTY NO: 102 MH6TYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNADTIGTARDYWGQGTLVTV SS SEQ ID MH6QVQLVESGGGVVQAGGSLRLSCAASGSTFSIRAMRWYRQAPGTERDLVAVIYGSSTY NO: 103(exemplary YADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNADTIGTARDYWGQGTLVTVhumanized SS version of 2A2) SEQ ID MH7QVQLVESGGGVVQPGGSLRLSCAASGSTFSIRAMRWYRQAPGKERELVAVIYGSSTY NO: 104(exemplary YADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNADTIGTARDYWGQGTLVTVhumanized SS version of 2A2) SEQ ID MH8QVQLVESGGGVVQPGGSLRLSCAASGSTFSIRAMRWVRQAPGKGLEWVSVIYGSSTY NO: 105(exemplary YADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNADTIGTARDYWGQGTLVTVhumanized SS version of 2A2)

Sequence Table for CDRs of exemplary Mesothelinbinding trispecific proteins of this disclosure Exemplary MSLN bindingSequence ID trispecific No. protein/TriTAC CDR1 Sequence 106 9B1GRTFSVRGMA 107 9F3 GSIPSIEQMG 108 7H2 GTTYTFDLMS 109 3B4 GSTSNINNMR 1104A2 GSTFGINAMG 111 12D1 ISAFRLMSVR 112 3G1 GRPFSINTMG 113 2A1 GSDFTEDAMA114 6F3 GSDFTEDAMA 115 1H2 GFTFSSFGMS 116 3F2 GLTYSIVAVG 117 2C2GLTFGVYGME 118 2D1 TTSSINSMS 119 6H2 GRTLSRYAMG 120 5D2 GSIFSPNAMI 1217C4 GATSAITNLG 122 5F2 GSTFRIRVMR 123 2C2 GDTSKFKAVG 124 5G2 GSTFGNKPMG125 9H2 GSTSSINTMY 126 5D4 GRTDRITTMG 127 2A4 GRTIGINDMA 128 7F1AIGSINSMS 129 5C2 GSTSSINTMY 130 2F4 TTFSINSMS 131 2A2 GSTFSIRAMR 13211F3 GRTSTIDTMY 133 10B3 GSTSSINTMY 134 MH1 GGDWSANFMY 135 MH2GGDWSANFMY 136 MH3 GSTSSINTMY 137 MH4 GSTSSINTMY 138 MHS GSTSSINTMY 139MH6 GSTFSIRAMR 140 MH7 GSTFSIRAMR 141 MH8 GSTFSIRAMR 142 MH9 GRTSTIDTMY143 MH10 GRTSTIDTMY 144 MH11 GRTSTIDTMY

Sequence Table for CDR2s of exemplary Mesothelinbinding trispecific proteins of this disclosure Exemplary trispecificSequence ID No. protein/TriTAC CDR2 Sequence 145 9B1 TMNPDGFPNYADAVKGRFT146 9F3 ALTSGGRANYADSVKGRFT 147 7H2 SISSDGRTSYADSVRGRFT 148 3B4VITRGGYAIYLDAVKGRFT 149 4A2 VISRGGSTNYADSVKGRFT 150 12D1TIDQLGRTNYADSVKGRFA 151 3G1 SISSSGDFTYTDSVKGRFT 152 2A1FVSKDGKRILYLDSVRGRFT 153 6F3 FVSKDGKRILYLDSVRGRFT 154 1H2SISGSGSDTLYADSVKGRFT 155 3F2 DISPVGNTNYADSVKGRFT 156 12C2SHTSTGYVYYRDSVKGRFT 157 2D1 VITDRGSTSYADSVKGRFT 158 6H2AISRSGGTTRYSDSVKGRFT 159 5D2 SINSSGSTNYGDSVKGRFT 160 7C4RISVREDKEDYEDSVKGRFT 161 5F2 VISGSSTYYADSVKGRFT 162 2C2WINNSGVGNTAESVKGRFT 163 5G2 VISSDGGSTRYAALVKGRFT 164 9H2FISSGGSTNVRDSVKGRFS 165 5D4 TISNRGTSNYANSVKGRFT 166 2A4TITKGGTTDYADSVDGRFT 167 7F1 VITDRGSTSYADSVKGRFT 168 5C2TINRGGSTNVRDSVKGRFS 169 2F4 VITNRGTTSYADSVKGRFT 170 2A2VIYGSSTYYADAVKGRFT 171 11F3 YVTSRGTSNVADSVKGRFT 172 10B3FISSGGSTNVRDSVKGRFS 173 MH1 RISGRGVVDYVESVKGRFT 174 MH2RISGRGVVDYVESVKGRFT 175 MH3 FISSGGSTNVRDSVKGRFT 176 MH4FISSGGSTNVRDSVKGRFT 177 MHS FISSGGSTNVRDSVKGRFT 178 MH6VIYGSSTYYADAVKGRFT 179 MH7 VIYGSSTYYADAVKGRFT 180 MH8 VIYGSSTYYADAVKGRFT181 MH9 YVTSRGTSNVADSVKGRFT 182 MH10 YVTSRGTSNVADSVKGRFT 183 MH11YVTSRGTSNVADSVKGRFT

Sequence Table for CDR3s of exemplary Mesothelinbinding trispecific proteins of this disclosure Exemplary Sequence IDtrispecific No. protein/TriTAC CDR3 Sequence 184 9B1 GPY 185 9F3GRFKGDYAQRSGMDY 186 7H2 QRSGVRAF 187 3B4 DRVEGTSGGPQLRDY 188 4A2RTYTRHDY 189 12D1 GGGPLGSRWLRGRH 190 3G1 RRTYLPRRFGS 191 2A1 APGAARNY192 6F3 APGAARNV 193 1H2 GGSLSRSS 194 3F2 VRGWLDERPGPGPIVY 195 12C2NRGSYEY 196 2D1 IADWRGY 197 6H2 RRRGWGRTLEY 198 5D2 SDFRRGTQY 199 7C4QRWGRGPGTT 200 5F2 DDSGIARDY 201 2C2 YRRFGINKNY 202 5G2 LRTYYLNDPVVFS203 9H2 YIPLRGTLHDY 204 5D4 RKWGRNY 205 2A4 KRREWAKDFEY 206 7F1 IADWRGY207 5C2 YIPYGGTLHDF 208 2F4 IADWRGY 209 2A2 DTIGTARDY 210 11F3 RTTSYPVDF211 10B3 YIPYGGTLHDF 212 MH1 ASY 213 MH2 ASY 214 MH3 YIPYGGTLHDF 215 MH4YIPYGGTLHDF 216 MHS YIPYGGTLHDF 217 MH6 DTIGTARDY 218 MH7 DTIGTARDY 219MH8 DTIGTARDY 220 MH9 RTTSYPVDF 221 MH10 RTTSYPVDF 222 MH11 RTTSYPVDF

Framework region 1 (f1) of exemplary MSLNtrispecific binding proteins of this disclosure Exemplary Sequencetrispecific ID No. protein/TriTAC Framework 1 223 9B1QVQLVESGGGLVQPGGSLRLSCAAS 224 9F3 QVQLVESGGGLVQAGGSLRLSCAAS 225 7H2QVQLVESGGGLVQAGGSLRLSCAFS 226 3B4 QVQLVESGGGLVQAGGSLRLSCVAS 227 4A2QVQLVESGGGLVQAGGSLRLSCAAS 228 12D1 QVRLVESGGGLVQAGGSLRLSCAAS 229 3G1QVRLVESGGGLVQAGESLRLSCAAS 230 2A1 QVQPVESGGGLVQPGGSLRLSCVVS 231 6F3QVQPVESGGGLVQPGGSLRLSCVVS 232 1H2 EVQLVESGGGLVQPGNSLRLSCAAS 233 3F2QVQIVESGGGLVQAGGSLRLSCVAS 234 12C2 QVQLVESGGGLVQTGGSLRLSCAAS 235 2D1QVQLVESGGGLVQAGGSLRLSCAAS 236 6H2 QVQLVESGGGLVQAGGSLRLSCAAS 237 5D2QVQLGESGGGLVQAGGSLRLSCAAS 238 7C4 QVQLVESGGGLVPSGGSLRLSCAAS 239 5F2QVQLVESGGGLVQAGGSLRLSCAAS 240 2C2 QVQLVESGGGLVQAGESRRLSCAVS 241 5G2QVQLVESGGGLVQAGGSLRLSCAAS 242 9H2 QVQLVESGGGLVQAGGSLRLSCAAS 243 5D4QVQLVESGGGLVQAGGSLRLSCVAS 244 2A4 QVQLVESGGGLVQARGSLRLSCTAS 245 7F1QVQLVESGGGLVQAGGSLRLSCAAS 246 5C2 QVQLVESGGGLVQAGGSLRLSCAAS 247 2F4QVQLVESGGGLVQAGGSLRLSCTTS 248 2A2 QVQLVESGGGLVQAGGSLTLSCAAS 249 11F3QVQLVESGGGLVQAGGSLRLSCVAS 250 10B3 QVQLVESGGGLVQAGGSLRLSCAAS 251 MH1EVQLVESGGGLVQPGGSLRLSCAAS 252 MH2 EVQLVESGGGLVQPGGSLRLSCAAS 253 MH3EVQLVESGGGLVQAGGSLRLSCAAS 254 MH4 EVQLVESGGGLVQPGGSLRLSCAAS 255 MH5EVQLVESGGGLVQPGGSLRLSCAAS 256 MH6 QVQLVESGGGVVQAGGSLRLSCAAS 257 MH7QVQLVESGGGVVQPGGSLRLSCAAS 258 MH8 QVQLVESGGGVVQPGGSLRLSCAAS 259 MH9EVQLVESGGGLVQAGGSLRLSCVAS 260 MH10 EVQLVESGGGLVQPGGSLRLSCAAS 261 MH11EVQLVESGGGLVQPGGSLRLSCAAS

Framework region 2 (f2) of exemplary MSLNtrispecific binding proteins of this disclosure Exemplary Sequencetrispecific ID No. protein/TriTAC Framework 2 262 9B1 WYRQAGNNRALVA 2639F3 WYRQAPGKQRELVA 264 7H2 WYRQAPGKQRTVVA 265 3B4 WYRQAPGKERELVA 266 4A2WYRQAPGKQRELVA 267 12D1 WYRQDPSKQREWVA 268 3G1 WYRQAPGKQRELVA 269 2A1WYRQASGKERESVA 270 6F3 WYRQASGKERESVA 271 1H2 WVRQAPGKGLEWVS 272 3F2WYRQAPGKEREMVA 273 12C2 WFRQAPGKQREWVA 274 2D1 WYRQAQGKQREPVA 275 6H2WFRQAPGKERQFVA 276 5D2 WHRQAPGKQREPVA 277 7C4 WYRRAPGQVREMVA 278 5F2WYRQAPGTERDLVA 279 2C2 WYRQAPGAQRELLA 280 5G2 WYRQAPGKQRELVA 281 9H2WYRQAPGKERELVA 282 5D4 WYRQAPGKQRELVA 283 2A4 WYRQAPGNQRELVA 284 7F1WYRQAPGKQREPVA 285 5C2 WFRQAPGEERELVA 286 2F4 WYRQAPGNQREPVA 287 2A2WYRQAPGTERDLVA 288 11F3 WHRQAPGNERELVA 289 10B3 WYRQAPGKERELVA 290 MH1WYRQAPGKQRELVA 291 MH2 WVRQAPGKGLEWVS 292 MH3 WYRQAPGKERELVA 293 MH4WYRQAPGKERELVA 294 MH5 WVRQAPGKGLEWVS 295 MH6 WYRQAPGTERDLVA 296 MH7WYRQAPGKERELVA 297 MH8 WVRQAPGKGLEWVS 298 MH9 WHRQAPGNERELVA 299 MH10WHRQAPGKERELVA 300 MH11 WVRQAPGKGLEWVS

Framework region 3 (f3) of exemplary MSLNtrispecific binding proteins of this disclosure Exemplary triSpecificSequence protein/ ID No. TriTAC Framework 3 301 9B1ISWDIAENTVYLQMNSLNSEDTTVYYCNS 302 9F3 ISGDNVRNMVYLQMNSLKPEDTAIYYCSA 3037H2 ISGENGKNTVYLQMNSLKLEDTAVYYCLG 304 3B4 ISRDNANNAIYLEMNSLKPEDTAVYVCNA305 4A2 ISRDNAENTVSLQMNTLKPEDTAVYFCNA 306 12D1ISKDSTRNTVYLQMNMLRPEDTAVYYCNA 307 3G1 ISRDNAKNTVYLQMNSLKPEDTAVYYCNA 3082A1 ISRDIDKKTVYLQMDNLKPEDTGVYYCNS 309 6F3 ISRDIYKKTVYLQMDNLKPEDTGVYYCNS310 1H2 ISRDNAKTTLYLQMNSLRPEDTAVYYCTI 311 3F2ISKENAKNTVYLQMNSLKPEDTAVYYCHI 312 12C2 ISRDNAKSTVYLQMNSLKPEDTAIYYCKA 3132D1 ISRDNAKNTVYLQMNSLKPEDTAIYTCHV 314 6H2 ISRDNAANTFYLQMNNLRPDDTAVYYCNV315 5D2 VSRDIVKNTMYLQMNSLKPEDTAVYYCSY 316 7C4ISRDNTQNLVYLQMNNLQPHDTAIYYCGA 317 5F2 ISRDNAKNTLYLQMNNLKPEDTAVYYCNA 3182C2 ISRDNAKNTVYLQMNRLTPEDTDVYYCRF 319 5G2 ISRDNAKNTVYLQMESLVAEDTAVYYCNA320 9H2 VSRDSAKNIVYLQMNSLTPEDTAVYYCNT 321 5D4ISRDNAKNTVYLQMNSLKPEDTAVYYCNA 322 2A4 ISRDNAKNTVYLQMNSLKPEDTAVYYCNT 3237F1 ISRDNAKNTVYLQMNSLKPEDTAIYTCHV 324 5C2 VSRDSAKNIVYLQMNRLKPEDTAVYYCNT325 2F4 ISRDNARNTVYLQMDSLKPEDTAIYTCHV 326 2A2ISRDNAKNTLYLQMNNLKPEDTAVYYCNA 327 11F3 ISRDNAKNTAYLQMNSLKPEDTAVYYCSV 32810B3 VSRDSAKNIVYLQMNSLKPEDTAVYYCNT 329 MH1 ISRDNSKNTLYLQMNSLRAEDTAVYYCAV330 MH2 ISRDNSKNTLYLQMNSLRAEDTAVYYCAV 331 MH3ISRDNSKNTLYLQMNSLRAEDTAVYYCNT 332 MH4 ISRDNSKNTLYLQMNSLRAEDTAVYYCNT 333MHS ISRDNSKNTLYLQMNSLRAEDTAVYYCNT 334 MH6 ISRDNSKNTLYLQMNSLRAEDTAVYYCNA335 MH7 ISRDNSKNTLYLQMNSLRAEDTAVYYCNA 336 MH8ISRDNSKNTLYLQMNSLRAEDTAVYYCNA 337 MH9 ISRDNSKNTLYLQMNSLRAEDTAVYYCSV 338MH10 ISRDNSKNTLYLQMNSLRAEDTAVYYCSV 339 MH11ISRDNSKNTLYLQMNSLRAEDTAVYYCSV

Framework region 4 (f4) of exemplary MSLNtrispecific binding proteins of this disclosure Exemplary Sequencetrispecific ID No. protein/TriTAC Framework 4 340 9B1 WGQGTQVTVSS 3419F3 WGKGTLVTVSS 342 7H2 WGQGTQVTVSS 343 3B4 FGQGTQVTVSS 344 4A2WGQGTQVTVSS 345 12D1 WGQGTQVTVSS 346 3G1 WGQGTQVTVSS 347 2A1 WGQGTQVTVSS348 6F3 WGQGTQVTVSS 349 1H2 QGTLVTVSS 350 3F2 WGQGTQVTVSS 351 12C2WGQGTQVTVSS 352 2D1 WGQGTQVTVSS 353 6H2 WGQGTQVTVSS 354 5D2 WGQGTQVTVSS355 7C4 WGQGTQVTVSS 356 5F2 WGQGTQVTVSS 357 2C2 WGQGTQVTVSS 358 5G2WGQGTQVTVSS 359 9H2 WGQGTQVTVSS 360 5D4 WGQGTQVTVSS 361 2A4 WGQGTQVTVSS362 7F1 WGQGTQVTVSS 363 5C2 WGQGTQVTVSS 364 2F4 WGQGTQVTVSS 365 2A2WGQGTQVTVSS 366 11F3 WGQGTQVTVSS 367 10B3 WGQGTQVTVSS 368 MH1WGQGTLVTVSS 369 MH2 WGQGTLVTVSS 370 MH3 WGQGTLVTVSS 371 MH4 WGQGTLVTVSS372 MHS WGQGTLVTVSS 373 MH6 WGQGTLVTVSSGG 374 MH7 WGQGTLVTVSSGG 375 MH8WGQGTLVTVSSGG 376 MH9 WGQGTLVTVS 377 MH10 WGQGTLVTVSS 378 MH11WGQGTLVTVSS

What is claimed is:
 1. A mesothelin (MSLN) binding trispecific protein,wherein said protein comprises (a) a first domain (A) which specificallybinds to human CD3; (b) a second domain (B) which is a half-lifeextension domain; and (c) a third domain (C) which specifically binds toMSLN, wherein the domains are linked in the order H₂N-(A)-(C)—(B)—COOH,H₂N—(B)-(A)-(C)—COOH, H₂N—(C)—(B)-(A)-COOH, or by linkers L1 and L2. 2.The MSLN binding trispecific protein of claim 1, wherein the firstdomain comprises a variable light domain and variable heavy domain eachof which is capable of specifically binding to human CD3.
 3. The MSLNbinding trispecific protein of claim 1, wherein said first domain ishumanized.
 4. The MSLN binding trispecific protein of claim 1, whereinthe second domain binds albumin.
 5. The MSLN binding trispecific proteinof claim 1, wherein said second domain comprises a scFv, a variableheavy domain (VH), a variable light domain (VL), a peptide, a ligand, ora small molecule.
 6. The MSLN binding trispecific protein of claim 1,wherein said third domain comprises a VHH domain, a scFv, a VH domain, aVL domain, a non-Ig domain, a ligand, a knottin, or a small moleculeentity that specifically binds to MSLN.
 7. The MSLN binding trispecificprotein of claim 6, wherein said third domain comprises a VHH domain. 8.The MSLN binding trispecific protein of claim 7, wherein said VHH domaincomprises one or more conserved regions comprising a sequence identicalto or comprising one or more amino acid residue substitutions relativeto SEQ ID NO: 41, 42, 43, or
 44. 9. The MSLN binding trispecific proteinof claim 1, wherein said third domain comprises the following formula:f1-r1-f2-r2-f3-r3-f4 wherein, r1 is identical to or comprises one ormore amino acid residue substitutions relative to any one of SEQ IDNos.: 51, 56, and 106-144; r2 is identical to or comprises one or moreamino acid residue substitutions relative to any one of SEQ ID Nos.: 52,55, and 145-183; and r3 is identical to or comprises one or more aminoacid residue substitutions relative to any one of SEQ ID Nos.: 53, 56,and 184-222; and wherein f1, f2, f3 and f4 are framework residues. 10.The MSLN binding trispecific protein of claim 1, wherein said thirddomain comprises a sequence that is at least 80% identical to a sequenceselected from the group consisting of SEQ ID NOs: 1-29.
 11. The MSLNbinding trispecific protein of claim 1, wherein said third domain is ahumanized VHH domain.
 12. The MSLN binding trispecific protein of claim11, wherein said humanized VHH domain comprises (i) a stretch of aminoacids corresponding to SEQ ID NO: 45, (ii) a stretch of amino acidscorresponding to SEQ ID NO: 46, (iii) a stretch of amino acidscorresponding to SEQ ID NO: 47, (iv) a stretch of amino acidscorresponding to SEQ ID NO: 48, (v) a stretch of amino acidscorresponding to SEQ ID NO: 49, and (vi) a stretch of amino acidscorresponding to SEQ ID NO:
 50. 13. The MSLN binding trispecific proteinof claim 11, wherein said humanized VHH domain comprises the followingformula:f1-r1-f2-r2-f3-r3-f4 wherein, r1 is identical to or comprises one ormore amino acid residue substitutions relative to any one of SEQ IDNos.: 134-44; r2 is identical to or comprises one or more amino acidresidue substitutions relative to any one of SEQ ID Nos.: 173-183; andr3 is identical to or comprises one or more amino acid residuesubstitutions relative to SEQ ID Nos.: 212-222; and wherein f1, f2, f3and f4 are framework residues.
 14. The MSLN binding trispecific proteinof claim 11, wherein said humanized VHH domain comprises a sequenceselected from the group consisting of SEQ ID NOs: 30-40, and 102-105.15. The MSLN binding trispecific protein of claim 1, wherein said thirddomain binds to a human mesothelin protein comprising the sequence setforth as SEQ ID NO:
 57. 16. The MSLN binding trispecific protein ofclaim 15, wherein said epitope is located in region I, comprising aminoacid residues 296-390 of SEQ ID NO: 57, region II comprising amino acidresidue 391-486 of SEQ ID NO: 57, or region III comprising amino acidresidues 487-598 of SEQ ID NO:
 57. 17. The MSLN binding trispecificprotein of claim 1, wherein linkers L1 and L2 are each independentlyselected from (GS)_(n) (SEQ ID NO: 87), (GGS)_(n) (SEQ ID NO: 88),(GGGS)_(n) (SEQ ID NO: 89), (GGSG)_(n) (SEQ ID NO: 90), (GGSGG)_(n) (SEQID NO: 91), or (GGGGS)_(n) (SEQ ID NO: 92), wherein n is 1, 2, 3, 4, 5,6, 7, 8, 9, or
 10. 18. The MSLN binding trispecific protein of claim 1,wherein linkers L1 and L2 are each independently (GGGGS)₄ (SEQ ID NO:95) or (GGGGS)₃ (SEQ ID NO: 96).
 19. The MSLN binding trispecificprotein of claim 1, wherein the domains are linked in the orderH₂N—(C)—(B)-(A)-COOH.
 20. The MSLN binding trispecific protein of claim1, wherein the protein is less than about 80 kDa.
 21. The MSLN bindingtrispecific protein of claim 1, wherein said protein has an eliminationhalf-time of at least about 50 hours.
 22. The MSLN binding trispecificprotein of claim 1, wherein said protein has increased tissuepenetration as compared to an IgG to the same MSLN.
 23. The MSLN bindingtrispecific protein of claim 1, wherein said protein comprises asequence selected from the group consisting of SEQ ID NOs: 58-86, 98,100, and
 101. 24. The MSLN binding trispecific protein of claim 1,wherein said third domain comprises a CDR1 sequence comprising asequence as set forth in any one of SEQ ID Nos.: 51, 54, and 106-144.25. The MSLN binding trispecific protein of claim 1, wherein said thirddomain comprises a CDR2 sequence comprising a sequence as set forth inany one of SEQ ID Nos.: 52, 55, and 145-183.
 26. The MSLN bindingtrispecific protein of claim 1, wherein said third domain comprises aCDR2 sequence comprising a sequence as set forth in any one of SEQ IDNos.: 53, 56, and 184-222.
 27. A mesothelin (MSLN) binding trispecificprotein, comprising the sequence as set forth in SEQ ID NO:
 98. 28. Amethod for the treatment or amelioration of a proliferative disease, ora tumorous disease, comprising administration of a mesothelin bindingtrispecific protein comprising a sequence as set forth in any one of SEQID Nos.: 58-86, 98, 100, and
 101. 29. The method of claim 28, comprisingadministering the mesothelin binding trispecific protein at a dose of upto 10 mg/kg.
 30. The method of claim 29, wherein the protein isadministered once a week, twice per week, every other day, or everythree weeks.